# Patent

## Title
Biosynthetic genes and host cells for the synthesis of polyketide antibiotics and method of use

## Abstract

Three gene clusters that together encode albicidin biosynthesis, the complete gene DNA sequences, and the deduced protein sequences for the enzymes and methods for using the DNA sequences are disclosed and discussed as well as methods for plant protection and creating new antibiotics. The novel Albicidin family of antibiotics is disclosed and their structure deduced.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage application of International Patent Application No. PCT/US2003/33142 filed Oct. 17, 2003, which claims the benefit of U.S. Provisional patent application with Ser. No. 60/419,463, filed Oct. 18, 2002 the disclosures of which are hereby incorporated by reference in their entirety, including all nucleic acid sequences, amino acid sequences, chemical formulae, tables and figures.


TECHNICAL FIELD
The Sequence Listing for this application is labeled “seq-list-replace.txt” which was created on Jun. 26, 2008 and is 323 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.
The invention is in the field of genetic engineering, and in particular the isolation and expression of the biosynthetic genes that produce a family of antibiotics known generically as albicidins.
BACKGROUND OF THE INVENTION
U.S. Pat. No. 4,525,354 to Birch and Patil described a “non-peptide” antibiotic of M.W. “about 842” called “albicidin.” Albicidin is described as produced by culturing chlorosis-inducing strains of Xanthomonas albilineans isolated from diseased sugarcane, and mutants thereof. The antibiotic was isolated from the culture medium by adsorption on resin and was purified by gel filtration and High Performance Liquid Chromatography (HPLC). The chemical structure of this antibiotic was not determined and remained unknown, although the Birch and Patil patent disclosed spectral data for a fraction having antibiotic activity and the presence of approximately 38 carbon atoms and at least one COOH group.
Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes leaf scald disease of sugarcane (interspecific hybrids of Saccharum species) (Ricaud and Ryan, 1989; Rott and Davis, 2000). Leaf scald symptoms include chlorosis, necrosis, rapid wilting, and plant death. Chlorosis-inducing strains of the pathogen produce several toxic compounds. The major toxic component, named albicidin, inhibits chloroplast DNA replication, resulting in blocked chloroplast differentiation and chlorotic leaf streaks that are characteristic of the plant disease (Birch and Patil, 1983, 1985b, 1987a and 1987b). Several studies established that albicidin plays a key role in pathogenesis and especially in the development of disease symptoms (Wall and Birch, 1997; Zhang and Birch, 1997; Zhang et al., 1999; Birch, 2001).
The prior art indicates that albicidin inhibits prokaryotic DNA replication and is bactericidal to a range of gram-positive and gram-negative bacteria (Birch and Patil, 1985a). Albicidin is therefore of interest as a potential clinical antibiotic (Birch and Patil, 1985a). However, low yield of toxin production in X. albilineans has slowed down studies into the chemical structure of albicidin and its therapeutic application (Zhang et al., 1998). The chemical structure of this albicidin remains unknown, however this albicidin has been partially characterized as a non-peptide antibiotic with a molecular weight of about 842 that contains approximately 38 carbon atoms with three or four aromatic rings, at least one COOH group, two OCH3 groups, a trisubstituted double bond and a CN linkage (Birch and Patil, 1985a; Huang et al., 2001).
Molecular cloning and characterization of the genes governing the biosynthesis of albicidin is of considerable interest because such information provides approaches to engineer overproduction of albicidin, to characterize its chemical structure, to allow therapeutic applications and to clarify the relationship between toxin production and the ability to colonize sugarcane. Two similar mutagenesis and complementation studies have been conducted to identify the genetic basis of albicidin production in X. albilineans strains isolated in two different geographical locations, Australia and Florida.
One study of X. albilineans strain LS155 from Australia revealed that genes for albicidin biosynthesis and resistance span at least 69 kb (Wall and Birch, 1997). Subsequently, three genes required for albicidin biosynthesis were identified, cloned and sequenced from two Australian strains of X. albilineans (LS155 and Xa13): xabA, xabB and xabC (Huang et al., 2001; Huang et al. 2000a, 2000b). The xabB gene encodes a large protein with a predicted size of 525.6 kDa, with a modular architecture indicative of a multi functional polyketide synthase (PKS) linked to a nonribosomal peptide synthetase (NRPS) (Huang et al., 2001). The xabC gene, located immediately downstream from xabB, encodes an S-adenosyl-L-methionine (SAM)-dependent O-methyltransferase (Huang et al., 2000a). The xabA gene, located in another region of the genome, encodes a phosphopantetheinyl transferase required for post-translational activation of PKS and NRPS enzymes (Huang et al., 2000b).
These first results demonstrated that the albicidin biosynthesis apparatus is a PKS and/or NRPS system. Such systems assemble simple acyl-coenzyme A or amino acid monomers to produce polyketides and/or nonribosomal peptides (Marahiel et al., 1997; Cane, 1997; Cane and Walsh, 1999). These metabolites form very large classes of natural products that include numerous important pharmaceuticals, agrochemicals, and veterinary agents such as antibiotics, immunosuppressants, anti-cholesterolemics, as well as antitumor, antifungal and antiparasitic agents. Genetic studies of prokaryotic PKS and NRPS produced detailed information regarding the function and the organization of genes responsible for the biosynthesis of polyketides and nonribosomal peptides. Such knowledge, in turn, made it possible to produce combinations of PKS and NRPS genes from different microorganisms in order to produce novel antibiotics (McDaniel et al., 1999; Rodriguez and McDaniel, 2001; Pfeifer et al., 2001). Investigating the complete albicidin biosynthesis apparatus is therefore of great interest because such results may contribute to the knowledge as to how PKS and NRPS interact and how they might be manipulated to engineer novel molecules.
A second study with X. albilineans strain Xa23R1 from Florida revealed that at least two gene clusters, one spanning more than 48 kb, are involved in albicidin production (Rott et al., 1996). This conclusion was based on the following data: (I) fifty Xa23R1 mutants defective in albicidin production were isolated; (ii) a Xa23R1 genomic library of 845 clones, designated pALB1 to pALB845, was constructed; (iii) two overlapping DNA inserts of approximately 47 kb and 41 kb, from clones pALB540 and pALB571 respectively, complemented forty-five mutants and were supposed to contain a major gene cluster involved in albicidin production; (iv) a DNA insert of approximately 36 kb, from clone pALB639, complemented four of the five remaining mutants not complemented by pALB540 and pALB571, and was supposed to contain a second region involved in albicidin production; and (v) the remaining mutant, AM37, which was not complemented by any of the three cosmids pALB540, pALB571 and pALB639, was supposed to be mutated in a third region of the genome involved in albicidin production.
The DNA sequences of all of the genes required to produce the albicidin family of polyketide antibiotics, the expressed protein amino acid sequences of all of the genes, and the deduced structure of Albicidin have not been previously reported, although fragmentary sequences that include three of the biosynthetic genes have been reported. Identification of one albicidin gene, xabC, as a methyltransferase gene involved in albicidin biosynthesis is reported by Huang, G., Zhang, L. & Birch, R. G. (2000a, Gene 255, 327-333) and claimed as biologically active in producing a polyketide antibiotic in PCT WO 02/24736 A1. Identification of a second albicidin gene, xabA, as a phosphopantetheinyl transferase gene is reported by Huang, G., Zhang, L. and Birch, R. G. (2000b) Gene 258, 193-199 and claimed as biologically active in producing a polyketide antibiotic in PCT WO 02/24736 A1. Huang, G., Zhang, L. & Birch, R. G. (2001) Microbiology 147, 631-642, report a DNA sequence of xabB (GenBank accession # AF239749), a multi functional polyketide-peptide synthetase that may be essential for albicidin biosynthesis in Xanthomonas albilineans. This xabB gene is reported as full length by Birch in PCT WO 02/24736 A1 (their seq. ID #1) and claimed by Birch in PCT WO 02/24736 A1 as a biologically active polyketide synthase of 4,801 amino acids in length, enabling production of albicidin. However, the DNA sequence reported by Huang et al. (2001) in GenBank AF239749 and by Birch in PCT WO 02/24736 A1 (their seq. ID #1) appears to be incomplete and missing 6,234 bp of DNA sequence encoding 2,078 amino acids. The subject invention provides the complete DNA sequence of xabB (albI, our seq. 20) as 20,637 bp, encoding a biologically active polyketide synthase of 6,879 amino acids of in this application (our seq ID #26). Factors affecting biosynthesis by Xanthomonas albilineans of albicidins antibiotics and phytotoxins are discussed in J. Appl. Microbiol. 85, 1023-1028. and Wall, M. K. & Birch, R. G. (1997). Genes for albicidin biosynthesis and resistance span at least 69 kb in the genome of Xanthomonas albilineans. Lett. Appl. Microbiol. 24, 256-260. A gene from X. albilineans strain Xa13, designed AlbF, which confers high level albicidin resistance in Escherichia coli and which encodes a putative albicidin efflux pump, was directly submitted to Genbank by Bostock and Birch (Accession No. AF403709).
SUMMARY OF THE INVENTION
The present invention describes and characterizes the family of antibiotics that is produced by culturing chlorosis-inducing strains of X. albilineans and mutants thereof, together with the complete set of twenty biosynthetic genes capable of producing the unique and previously uncharacterized family of antibiotics produced by X. albilineans and previously lumped together as “albicidins.” The set of twenty biosynthetic genes isolated, purified and cloned from a culture of X. albilineans revealed that this set of biosynthetic genes is capable of synthesizing products exhibiting a high level of variation among the products, indicating that albicidins comprise a family of polyketide antibiotics. The albicidins described in the present invention are synthesized by twenty genes, including one polyketide-peptide synthase, one polyketide synthase and two peptide synthases, but the substrates of the polyketide-peptide synthase and of one peptide synthase are not α-amino acids. The biosynthetic enzymes represent a previously undescribed and unique polyketide antibiotic biosynthetic system.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a Physical Map and genetic organization of the DNA Region containing the major gene cluster XALB1 involved in the biosynthesis of Albicidins.
FIG. 2 is an illustration of the organization of the four PKS modules and the seven NRPS modules identified in cluster XALB1 and comparison with the organization of the prior art material XabB.
FIG. 3 shows the conserved sequence motifs in O-methyltransferases and C-methyltransferases involved in antibiotic biosynthesis in bacteria and in AlbII.
FIG. 4 shows the conserved sequence motifs in O-methyltransferases and in different tcmP-like hypothetical proteins and AlbVI.
FIG. 5 is an illustration of the alignment of the primary sequences between the conserved motifs A4 and A5 of Alb NPRSs and PKS-4 in Xanthomonas albilineans with the corresponding sequences of GrsA (Phe) accession number: P14687 SEQ ID NO: 132 and Blm NRPS-2 (β-Ala) accession number AF210249 (SEQ ID NO: 133); AlbI NRPS-1 (SEQ ID NO: 134); AlbI NRPS-3 (SEQ ID NO: 135); AlbIX NRPS-6 (SEQ ID NO: 136); AlbIX NRPS-7 (SEQ ID NO: 137); AlbIV NRPS-5 (SEQ ID NO: 138); AlbVII PKS-4 (SEQ ID NO: 139); AlbI NRPS-2 (SEQ ID NO: 140).
FIG. 6 shows Rho-independent transcription terminators identified in the intergenic regions of XALB1 and XALB3 clusters (SEQ ID NO: 141, XALB1 Strand+(29 bp downstream from the TGA stop codon of albXVII); SEQ ID NO: 142, XALB1 Strand+(400 bp downstream from the TAA stop codon of albIV); SEQ ID NOs: 143, 144 and 145, XALB1 Strand−(62 bp, 170 bp and 560 bp downstream from the TAG stop codon of albXVI); SEQ ID NOs: 146 and 147, XLAB3 Strand+).
FIG. 7A shows sequences identified as a putative bidirectional promoter between albX and albXVII in XALB1 for transcriptional control of operons 3 and 4 (SEQ ID NOs: 148 and 149).
FIG. 7B shows sequences identified as a putative unidirectional promoter upstream from albXIX for transcriptional control of operon 5 if albXVIII is not expressed (SEQ ID NOs: 152 and 153).
FIG. 8 is a physical map and genetic organization of the DNA region containing the gene clusters XALB2 and XALB3 involved in albicidin production.
FIG. 9A is linear model 1 leading to the biosynthesis of only one polyketide-polypeptide albicidin backbone.
FIG. 9B is linear model 2 leading to the biosynthesis of four different polyketide-polypeptide backbone.
FIG. 10A is an alignment of the conserved motifs in AT domains from RifA-1 (SEQ ID NO: 156), -2(SEQ ID NO: 157), -3 (SEQ ID NO: 158), RifB-1 (SEQ ID NO: 159), RitE-1 (SEQ ID NO: 160) (Rifamycin PKSs, August et al., 1998) and BlmVIII (Bleomycin PKS; Du et al., 2000) (SEQ ID NO: 161).
FIG. 10B is a comparison of AlbXIII (SEQ ID NO: 162), FenF (a malonyl-CoA transacylase located upstream from mycA, Duitman et al., 1999) (SEQ ID NO: 163) and LipA (a lipase; Valdez et al., 1999) (SEQ ID NO: 164).
FIG. 11A is a proposed model for biosynthesis of albicidin, including putative substrates of PKS and NRPS modules.
FIG. 11B shows the proposed compositions and structures of albicidins.
FIG. 12 illustrates subcloning of operons 3 and 4 (from pALB540), XALB2 (from pAC389.1) and XALB3 (from pEV639) into a single plasmid, pOp3-4/XALB2-3. A BamHI-PstI fragment from pALB540, corresponding to a portion of operon 4, was subcloned into pBCKS(+), yielding pBC/Op4D (step 1). A XhoI site was introduced into this vector immediately upstream from the BfrI site by directed mutagenesis, yielding pBC/Op4DXhoI (step 2). The EcoRI fragment from pAC389.1 (XALB2) was then subcloned into pBC/Op4DXhoI, yielding pBC/Op4D/XALB2 (step 3). A BfrI fragment from pALB540 containing complete operon 3 and the beginning of operon 4 was subcloned into pBC/Op4D/XALB2, yielding pBC/Op3-4/XALB2 (step 4). The SalI fragment from pEV639 (XALB3) was subcloned into pBKS, yielding pBKS/XALB3 (step 5). The SalI site located on the KpnI side of the polylinker was then destroyed and substituted by a XhoI restriction site, yielding pBKS/XALB3XhoI (step 6). Finally, the XhoI cassette of pBC/Op3-4/XALB2 was subcloned into the SalI restriction site of pBKS/XALB3XhoI, yielding pBKS/Op3-4/XALB2-3 (step 7). An XhoI site was added to the BamHI site of pLAFR3, yielding pLAFR3XhoI (step 8). The XhoI cassette from pBKS/Op3-4/XALB2-3 was then cloned into pLAFR3XhoI, yielding pOp3-4/XALB2-3 (step 9).



DETAILED DESCRIPTION OF THE INVENTION
The invention results from the DNA sequencing of the complete major gene cluster XALB1, as well as the noncontiguous fragments XALB2 and XALB3. XALB1 is present in the two overlapping DNA inserts of clones pALB540 and pALB571. Reading frame analysis and homology analyses allow one to predict the genetic organization of XALB1 and to assign a function to the genes potentially required for albicidin production. Based on the alignment of the different PKS and/or NRPS enzymes encoded by XALB1 we proposed a model for the albicidin backbone biosynthesis. However the invention disclosed herein does not depend upon the accuracy of the proposed model. The invention includes the successful cloning and DNA sequencing of the second region of the genome (XALB2) involved in albicidin production and mutated in mutant AM37.
The invention includes the characterization of the third region of the genome (XALB3) involved in albicidin production present in clone pALB639. These results allowed the possibility to characterize all enzymes of the albicidin biosynthesis pathway including structural, resistance and regulatory elements and to engineer overproduction of albicidin.
The subject invention provides:

    
    
        (a) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25;
        (b) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47;
        (c) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide that is complementary to a polynucleotide selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25;
        (d) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide that is complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 26, 27; 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47; or
        (e) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide that is at least 70% homologous to: (1) a polynucleotide selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25; (2) a polynucleotide sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47; (3) a polynucleotide that is complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47; (3) a polynucleotide that is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25;
        (f) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide sequence encoding variant (e.g., a variant polypeptide) of a polypeptide selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47, wherein said variant has at least on of the biological activities associated with the polypeptides of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47;
        g) isolated, recombinant, and/or purified polynucleotide sequences comprising polynucleotide sequence encoding a fragment of a polypeptide selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47 or a fragment of a variant polypeptide of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47;
        h) isolated, recombinant, and/or purified polynucleotide sequences comprising a polynucleotide sequence encoding multimeric construct;
        j) a genetic construct comprising a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h);
        k) a vector comprising a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h);
        l) a host cell comprising a vector a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h);
        m) a transformed plant cell comprising a vector comprising a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h);
        n) a transformed plant comprising a vector comprising a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h); or;
        o) a polynucleotide that hybridizes under low, intermediate or high stringency with a polynucleotide sequence as set forth in (a), (b), (c), (d), (e), (f), (g), or (h).
    
    


“Nucleotide sequence”, “polynucleotide” or “nucleic acid” can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules). It should also be understood that the present invention does not relate to genomic polynucleotide sequences in their natural environment or natural state. The nucleic acid, polynucleotide, or nucleotide sequences of the invention can be isolated, purified (or partially purified), by separation methods including, but not limited to, ion-exchange chromatography, molecular size exclusion chromatography, or by genetic engineering methods such as amplification, subtractive hybridization, cloning, subcloning or chemical synthesis, or combinations of these genetic engineering methods.
A homologous polynucleotide or polypeptide sequence, for the purposes of the present invention, encompasses a sequence having a percentage identity with the polynucleotide or polypeptide sequences, set forth herein, of between at least (or at least about) 70.00% to 99.99% (inclusive). The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 20.00% and, up to, including 99.99%. These percentages are purely statistical and differences between two nucleic acid sequences can be distributed randomly and over the entire sequence length. For example, homologous sequences can exhibit a percent identity of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent with the sequences of the instant invention. Typically, the percent identity is calculated with reference to the polynucleotide of a particular SEQ ID NO.; the full-length of a selected polynucleotide, or the native (naturally occurring) polynucleotide. The terms “identical” or percent “identity”, in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
A “complementary” polynucleotide sequence, as used herein, generally refers to a sequence arising from the hydrogen bonding between a particular purine and a particular pyrimidine in double-stranded nucleic acid molecules (DNA-DNA, DNA-RNA, or RNA-RNA). The major specific pairings are guanine with cytosine and adenine with thymine or uracil. A “complementary” polynucleotide sequence may also be referred to as an “antisense” polynucleotide sequence or an “antisense” sequence.
Sequence homology and sequence identity can also be determined by hybridization studies under high stringency, intermediate stringency, and/or low stringency. Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under low, intermediate, or high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak [1987] DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
It is also well known in the art that restriction enzymes can be used to obtain functional fragments of the subject DNA sequences. For example, Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA (commonly referred to as “erase-a-base” procedures). See, for example, Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Wei et al. [1983] J. Biol. Chem. 258:13006-13512.
The present invention further comprises fragments of the polynucleotide sequences of the instant invention. Representative fragments of the polynucleotide sequences according to the invention will be understood to mean any nucleotide fragment having at least 5 successive nucleotides, preferably at least 12 successive nucleotides, and still more preferably at least 15 or at least 20 successive nucleotides of the sequence from which it is derived. The upper limit for such fragments is the total number of nucleotides found in the full-length sequence encoding a particular polypeptide (e.g., a polypeptide selected from the group consisting of SEQ ID NOs: 26-50). The term “successive” can be interchanged with the term “consecutive”. In some embodiments, a polynucleotide fragment may be referred to as “a contiguous span of at least X nucleotides, wherein X is any integer value beginning with 5. The upper limit for polynucleotide fragments of the subject invention is the total number of nucleotides found in the full-length sequence of a particular SEQ ID or the total number of nucleotides encoding a particular polypeptide (e.g., a particular SEQ ID NO).
In some embodiments, the subject invention includes those fragments capable of hybridizing under various conditions of stringency conditions (e.g., high or intermediate or low stringency) with a nucleotide sequence according to the invention; fragments that hybridize with a nucleotide sequence of the subject invention can be, optionally, labeled as set forth below.
Thus, the subject invention also provides detection probes (e.g., fragments of the disclosed polynucleotide sequences) for hybridization with a target sequence or the amplicon generated from the target sequence. Such a detection probe will comprise a contiguous/consecutive span of at least 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Labeled probes or primers are labeled with a radioactive compound or with another type of label as set forth above. Alternatively, non-labeled nucleotide sequences may be used directly as probes or primers; however, the sequences are generally labeled with a radioactive element (32P, 35S, 3H, 125I) or with a molecule such as biotin, acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine, or fluorescein to provide probes that can be used in numerous applications.
The subject invention also provides for modified nucleotide sequences. Modified nucleic acid sequences will be understood to mean any nucleotide sequence that has been modified, according to techniques well known to persons skilled in the art, and exhibiting modifications in relation to the native, naturally occurring nucleotide sequences.
The subject invention also provides genetic constructs comprising: a) a polynucleotide sequence encoding a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1-25; b) a polynucleotide sequence having at least about 70% to 99.99% identity to a polynucleotide sequence encoding a polypeptide sequence selected from the group consisting of SEQ ID NO: 26-50, wherein said polynucleotide encodes a polypeptide having at least one of the biological activities of the polypeptides (e.g., a catalytic activity as set forth in Table 4); c) a polynucleotide sequence encoding a biologically active fragment of a polypeptide selected from the group consisting of SEQ ID NO: 26-50, wherein said biologically active fragment has at least one of the biological activities of the polypeptides (e.g., a catalytic or transport activity as set forth in Table 4); d) a polynucleotide sequence comprising SEQ ID NO: 1, 2, 3, or combinations thereof; e) a polynucleotide sequence encoding variant (e.g., a variant polypeptide) of a polypeptide selected from the group consisting of SEQ ID NOs: 2648, wherein said variant has at least on of the biological activities associated with the polypeptides (e.g., a catalytic or transport activity as set forth in Table 4); f) a polynucleotide sequence encoding a fragment of a variant polypeptide as set forth in (e); or g) a polynucleotide sequence encoding multimeric construct. Genetic constructs of the subject invention can also contain additional regulatory elements such as promoters and enhancers and, optionally, selectable markers.
Also within the scope of the subject instant invention are vectors or expression cassettes containing polynucleotides encoding the polypeptides, set forth supra, operably linked to regulatory elements. The vectors and expression cassettes may contain additional transcriptional control sequences as well. The vectors and expression cassettes may further comprise selectable markers. The expression cassette may contain at least one additional gene, operably linked to control elements, to be co-transformed into the organism. Alternatively, the additional gene(s) and control element(s) can be provided on multiple expression cassettes. Such expression cassettes are provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette(s) may additionally contain selectable marker genes operably linked to control elements.
In some embodiments, the expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the invention, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, may be native or analogous, or foreign or heterologous, to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
Where appropriate, the polynucleotides encoding the polypeptides set forth supra can be optimized for expression in the transformed plant That is, the genes can be synthesized using plant-preferred codons corresponding to the plant of interest Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
The expression cassettes may additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region), Elroy-Stein et al. (1989) PNAS USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMV Leader (Maize Dwarf Mosaic Virus), Virology 154:9-20; human immunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature 325:622-625; tobacco mosaic virus leader (TMV), Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256; and maize chlorotic mottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized.
Also provided are transformed host cells, transformed plant cells and transgenic plants which contain one or more genetic constructs, vectors, or expression cassettes comprising polynucleotides of the subject invention, or biologically active fragments thereof, operably linked to control elements. As used herein, the term “planta” includes algae and higher plants. Thus, algae, monocots, and dicots may be transformed with genetic constructs of the invention, expression cassettes, or vectors according to the invention. In certain embodiments of the subject invention, the transformed cells or transgenic plants comprise at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-25. In certain preferred embodiments, transformed cells or transgenic plants comprise at least one polynucleotide sequence comprising SEQ ID NOs: 1, 2, or 3. Optionally, the transformed cells or transgenic plants can comprise at least two or all three polynucleotide sequences selected from the group consisting of SEQ ID NOs: 1, 2, and 3.
Methods of transforming cells with genetic constructs, vectors, or expression cassettes comprising the novel polynucleotides of the invention are also provided. These methods comprise transforming a plant or plant cell with a polynucleotide according to the subject invention. Plants and plant cells may be transformed by electroporation, Agrobacterium transformation (including vacuum infiltration), engineered plant virus replicons, electrophoresis, microinjection, micro-projectile bombardment, vacuum infiltration of Agrobacterium, micro-LASER beam-induced perforation of cell wall, or simply by incubation with or without polyethylene glycol (PEG). Plants transformed with a genetic construct of the invention may be produced by standard techniques known in the art for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transferability. Agrobacterium transformation is used by those skilled in the art to transform algae and dicotyledonous species. Substantial progress has been made towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants. In particular, Agrobacterium mediated transformation has now emerged as a highly efficient transformation method in monocots. Microprojectile bombardment, electroporation, and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g., bombardment with Agrobacterium-coated microparticles (EP-A486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g., from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant. Available techniques are reviewed in Vasil et al. (1984) in Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and II, Laboratory Procedures and Their Applications (Academic press); and Weissbach et al. (1989) Methods for Plant Mol. Biol.
The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
Also according to the invention, there is provided a plant cell having the constructs of the invention. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. For integration of the construct into the plant genome, such introduction will be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. RNA encoded by the introduced nucleic acid construct may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such descendants should show the desired phenotype.
The present invention also provides a plant comprising a plant cell as disclosed. Transformed seeds and plant parts are also encompassed. As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to naturally occurring, deliberate, or inadvertent caused mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
In addition to a plant, the present invention provides any clone of such a plant, seed, or hybrid descendants, and any part of any of these, such as cuttings or seed. The invention provides any plant propagule that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed, and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone, or descendant of such a plant; or any part or propagule of said plant, off-spring, clone, or descendant. Plant extracts and derivatives are also provided.
As is apparent to the routineer in this technology, the disclosed methods allow for the expression of a gene of interest in any plant. The invention thus relates generally to methods for the production of transgenic plants (both monocots and dicots). As used herein, the term “transgenic plants” refers to plants (algae, monocots, or dicots), comprising plant cells in which homologous or heterologous polynucleotides are expressed as the result of manipulation by the hand of man.
As is apparent to one of ordinary skill in the art, the peptides encoded by the disclosed herein may be encoded by multiple polynucleotide sequences because of the redundancy of the genetic code. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, amino acid sequences. These variant DNA sequences are within the scope of the subject invention.
The terms “purified” and “isolated”, when referring to a polynucleotide, nucleotide, or nucleic acid, indicate a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecules but is not flanked by both of the coding or non-coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs (e.g., DNA excised with a restriction enzyme); (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transfected cells, and (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term “polynucleotide” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule and thus includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
“Control elements” include both “transcriptional control elements” and “translational control elements”. “Transcriptional control elements” include “promoter”, “enhancer”, and “transcription termination” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis et al. [1987] Science 236:1237). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plants, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the peptide of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types [for review see Voss et al. [1986] Trends Biochem. Sci. 11:287 and Maniatis et al. [1987] supra. Transcriptional control elements suitable for use in plants are well known in the art. “Translational control elements” include translational initiation regions and translational termination regions functional in plants.
A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. Strong promoters may be used to produce high levels of gene transcription. Alternatively, inducible promoters may be used to selectively active gene transcription when the appropriate signal is provided. Constitutive promoters may be utilized to continuously drive gene transcription. Tissue specific promoters may also be used in the practice of the invention in order to provide localized production of gene transcripts in a desired tissue. Developmental promoters may, likewise, be used to drive transcription of a gene during a particular developmental stage of the plant. Thus, a gene of interest can be combined with constitutive, tissue-specific, inducible, developmental, or other promoters for expression in plants depending upon the desired outcome.
Constitutive promoters include, for example, CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812; rice actin (McElroy et al. (1990) Plant Cell 2:163-171; ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. Each of the aforementioned patents and references is hereby incorporated by reference in its entirety.
A number of inducible promoters are known in the art. For example, a pathogen-inducible promoter can be utilized. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116; Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386; Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200; each of which is incorporated by reference in its entirety.
Wound-inducible promoters may be used in the genetic constructs of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498; wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150; and the like. These references are also incorporated by reference in their entireties.
Tissue specific promoters can also be used in the practice of the subject invention. For example, leaf-specific promoters can similarly be used if desired, and are taught in references which include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russel et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5)773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci USA:90(20) 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Alternatively, root-specific promoters are known and can be selected from the many available from the literature. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). Bogusz et al. (1990) Plant Cell 2(7):633-641 (root specific promoters from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomeniosa; Leach and Aoyagi (1991) Plant Science (Limerick) 79(1):69-76 (rolC and rolD root-including genes of Agrobacterium rhizogenes); Teeri et al. (1989) EMBO J. 8(2):343-350 (octopine synthase and TR2′ gene); (VfENOD-GRP3 gene promoter); Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772 and Capana et al. (1994) Plant Mol. Biol. 25(4):681-691 (rolB promoter). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
Other tissue specific promoters can also be used in the practice of the subject invention (see, for example U.S. Pat. No. 6,544,783). For example, xylem/vascular/tracheid-specific promoters, such as those disclosed in Milioni et al. (2002) Plant Cell, 14:2813-2824; Zhong et al. (1999) Plant Cell, 11:2139-2152; Ito et al. (2002) Plant Cell, 14:3201-3211; Parker et al. (2003) Development 130:2139-2148; Bourquin et al. (2002) Plant Cell 14:3073-3088 (each of which is hereby incorporated by reference in its entirety) can be used in the practice of the subject invention.
“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) Bioassays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ10B1 (Maize 19 kDa zein); celA (cellulose synthase); gama-zein; Glob-1; bean β-phaseolin; napin; β-conglycinin; soybean lectin; cruciferin; maize 15 kDa zein; 22 kDa zein; 27 kDa zein; g-zein; waxy; shrunken 1; shrunken 2; globulin 1; etc.
“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
As used herein, the term “expression cassette” refers to a molecule comprising at least one coding sequence operably linked to a control sequence which includes all nucleotide sequences required for the transcription of cloned copies of the coding sequence and the translation of the mRNAs in an appropriate host cell. Such expression cassettes can be used to express eukaryotic genes in a variety of hosts such as bacteria, green algae, cyanobacteria, plant cells, fungal cells, yeast cells, insect cells and animal cells. Under the invention, expression cassettes can include, but are not limited to, cloning vectors, specifically designed plasmids, viruses or virus particles. The cassettes may further include an origin of replication for autonomous replication in host cells, selectable markers, various restriction sites, a potential for high copy number and strong promoters.
By “vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
During the preparation of the constructs, the various fragments of DNA will often be cloned in an appropriate cloning vector, which allows for amplification of the DNA, modification of the DNA or manipulation of the DNA by joining or removing sequences, linkers, or the like. Preferably, the vectors will be capable of replication to at least a relatively high copy number in E. coli. A number of vectors are readily available for cloning, including such vectors as pBR322, vectors of the pUC series, the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla, Calif.).
In order to provide a means of selecting transformed plants or plant cells, the vectors for transformation will typically contain a selectable marker gene. Marker genes are expressible DNA sequences which express a polypeptide which resists a natural inhibition by, attenuates, or inactivates a selective substance. Examples of such substances include antibiotics and, in the case of plant cells, herbicides. Selectable markers for use in animal, bacterial, plant, fungal, yeast, and insect cells are well known in the art. Exemplary selectable markers include bacterial transposons Tn5 or Tn 601(903) conferring resistance to aminoglycosides (selection for Geneticin-resistance (G418R), mycophenolic acid resistance (MPAR) (utilizing E. coli guanosine phosphoribosyl transferase (gpt) encoding the enzyme XGPRT; selection is performed on medium containing MPA and xanthin), methotrexate resistance (MTXR), or cadmium-resistance which incorporates the mouse metallotheionein gene (as cDNA cassette) on the vector which detoxifies heavy metal ions by chelating them.
Alternatively, a marker gene may provide some visible indication of cell transformation. For example, it may cause a distinctive appearance or growth pattern relative to plants or plant cells not expressing the selectable marker gene in the presence of some substance, either as applied directly to the plant or plant cells or as present in the plant or plant cell growth media. The use of such a marker for identification of plant cells containing a plastid construct has been described (Svab et al. [1993] supra). Numerous additional promoter regions may also be used to drive expression of the selectable marker gene, including various plant promoters and bacterial promoters which have been shown to function in plants.
A number of other markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes which encode a product involved in chloroplast metabolism may also be used as selectable markers. For example, genes which provide resistance to plant herbicides such as glyphosate, bromoxynil or imidazolinone may find particular use. Such genes have been reported (Stalker et al. [1985] J. Biol. Chem. 260:4724-4728 (glyphosate resistant EPSP); Stalker et al. [1985] J. Biol. Chem. 263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan et al. [1990] Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene)).
Another aspect of the invention provides vectors for the cloning and/or the expression of a polynucleotide sequences taught herein in procaryotic or animal cells. The subject invention also provides for the expression of a polypeptide, peptide, derivative, or variant encoded by a polynucleotide sequence disclosed herein comprising the culture of a procaryotic or animal cell (a host cell) transformed with a polynucleotide of the subject invention under conditions that allow for the expression of a polypeptide, biologically active fragment, or multimeric construct encoded by said polynucleotide and, optionally, recovering the expressed polypeptide, peptide, derivative, or analog.
In this aspect of the invention, the polynucleotide sequences can be regulated by a second nucleic acid sequence so that the protein or peptide is expressed in a host cell transformed with the recombinant DNA molecule. For example, expression of a protein or peptide may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression include, but are not limited to, the CMV-IE promoter, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes simplex thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic vectors containing promoters such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., 1983, Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, and/or the alkaline phosphatase promoter.
The vectors according to the invention are, for example, vectors of plasmid or viral origin. In a specific embodiment, a vector is used that comprises a promoter operably linked to a nucleic acid sequence encoding a polypeptide as disclosed herein, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Expression vectors comprise regulatory sequences that control gene expression, including gene expression in a desired host cell. Exemplary vectors for the expression of the polypeptides of the invention include the pET-type plasmid vectors (Promega) or pBAD plasmid vectors (Invitrogen) or those provided in the examples below. Furthermore, the vectors according to the invention are useful for transforming host cells so as to clone or express the polynucleotide sequences of the invention.
The invention also encompasses the host cells transformed by a vector according to the invention. These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the polynucleotide sequences of the subject invention.
The host cell may be chosen from eukaryotic or prokaryotic systems, such as for example bacterial cells, (Gram negative or Gram positive), yeast cells (for example, Saccharomyces cereviseae or Pichia pastoris), animal cells (such as Chinese hamster ovary (CHO) cells), plant cells (e.g., algae), and/or insect cells using baculovirus vectors. In some embodiments, the host cells for expression of the polypeptides include, and are not limited to, those taught in U.S. Pat. No. 6,319,691, 6,277,375, 5,643,570, or 5,565,335, each of which is incorporated by reference in its entirety, including all references cited within each respective patent.
Furthermore, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can also to provide glycosylation of a protein.
The subject invention provides one or more isolated polypeptides comprising:
(a) SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47;
(b) a heterologous polypeptide sequence fused, in frame, to a polypeptide comprising SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47;
(c) a fragment of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, wherein said fragment exhibits at least one biological function of the polypeptide of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47; or
(d) a variant having at least 70% homology to a polypeptide comprising SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, wherein said variant exhibits at least one biological function of the polypeptide comprising SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47.
The term “peptide” may be used interchangeably with “oligopeptide” or “polypeptide” in the instant specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Linker elements can be joined to the polypeptides of the subject invention through peptide bonds or via chemical bonds (e.g., heterobifunctional chemical linker elements).
The subject invention encompasses polypeptide fragments of the full-length polypeptides disclosed herein. Polypeptide fragments, according to the subject invention, usually comprise a contiguous span of at least 5 consecutive (or contiguous) amino acids. The maximum length for a polypeptide fragment in the context of this invention is an integer that is one amino acid less than the full length of a particular SEQ ID NO: from which the fragment was derived. In certain preferred embodiments, fragments of the polypeptides of the subject invention retain at least one biological activity/function of the full-length polypeptide from which they are derived (e.g., such similar or identical enzymatic activity or the ability to provide resistance to an antibiotic or transport an antibiotic out of a cell (see, for example, Table 4).
A “variant” polypeptide (or polypeptide variant) is to be understood to designate polypeptides exhibiting, in relation to the natural polypeptide, certain modifications. These modifications can include a deletion, addition, or substitution of at least one amino acid, a truncation, an extension, a chimeric fusion, a mutation, or polypeptides exhibiting post-translational modifications. Among the homologous polypeptides, those whose amino acid sequences exhibit between at least (or at least about) 70.00% to 99.99% (inclusive) identity to the full length, native, or naturally occurring polypeptide are another aspect of the invention. The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 70.00% and, up to, including 99.99%. These percentages are purely statistical and differences between two polypeptide sequences can be distributed randomly and over the entire sequence length. Thus, variant polypeptides can have 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the polypeptide sequences of the instant invention. In certain preferred embodiments, variants of the polypeptides of the subject invention retain at least one biological activity/function of the full-length polypeptide from which they are derived (e.g., such as similar or identical enzymatic activity or the ability to provide resistance to an antibiotic or transport an antibiotic out of a cell (see, for example, Table 4).
Variant polypeptides can also comprise one or more heterologous polypeptide sequences (e.g., tags that facilitate purification of the polypeptides of the invention (see, for example, U.S. Pat. No. 6,342,362, hereby incorporated by reference in its entirety; Altendorf et al. [1999-WWW, 2000] “Structure and Function of the Fo Complex of the ATP Synthase from Escherichia Coli,” J. of Experimental Biology 203:19-28, The Co. of Biologists, Ltd., G. B.; Baneyx [1999] “Recombinant Protein Expression in Escherichia coli,” Biotechnology 10:411-21, Elsevier Science Ltd.; Eihauer et al. [2001] “The FLAGTM Peptide, a Versatile Fusion Tag for the Purification of Recombinant Proteins,” J. Biochem Biophys Methods 49:455-65; Jones et al. J. Chromatography 707:3-22; Jones et al. [1995] “Current Trends in Molecular Recognition and Bioseparation,” J. of Chromatography A. 707:3-22, Elsevier Science B. V.; Margolin [2000] “Green Fluorescent Protein as a Reporter for Macromolecular Localization in Bacterial Cells,” Methods 20:62-72, Academic Press; Puig et al. [2001] “The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification,” Methods 24:218-29, Academic Press; Sassenfeld [1990] “Engineering Proteins for Purification,” TibTech 8:88-93; Sheibani [1999] “Prokaryotic Gene Fusion Expression Systems and Their Use in Structural and Functional Studies of Proteins,” Prep. Biochem. & Biotechnol. 29(1):77-90, Marcel Dekker, Inc.; Skerra et al. [1999] “Applications of a Peptide Ligand for Streptavidin: the Strep-tag”, Biomolecular Engineering 16:79-86, Elsevier Science, B.V.; Smith [1998] “Cookbook for Eukaryotic Protein Expression: Yeast, Insect, and Plant Expression Systems,” The Scientist 12(22):20; Smyth et al. [2000] “Eukaryotic Expression and Purification of Recombinant Extracellular Matrix Proteins Carrying the Strep II Tag”, Methods in Molecular Biology, 139:49-57; Unger [1997] “Show Me the Money: Prokaryotic Expression Vectors and Purification Systems,” The Scientist 11(17):20, each of which is hereby incorporated by reference in their entireties), or commercially available tags from vendors such as such as STRATAGENE (La Jolla, Calif.), NOVAGEN (Madison, Wis.), QIAGEN, Inc., (Valencia, Calif.), or InVitrogen (San Diego, Calif.). Alternatively, the heterologous sequences may provide for the multimerization of the polypeptides of the subject invention (see, e.g., U.S. Pat. No. 5,478,925, WO 98/49305, or U.S. Pat. No. 5,073,627, Landschulz et al., (1988), Science. 240:1759, WO 94/10308, Hoppe et al., (1994), FEBS Letters. 344:191). Other methods of making multimers include the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide using techniques known in the art. Where biotin is attached to a polypeptide, avidin can be utilized to create multimers of the polypeptides to which the biotin element is attached (see, e.g., U.S. Pat. No. 5,478,925 for numerous methods of multimerization). Multimers of the invention may also be generated using chemical or genetic engineering techniques known in the art
The invention, thus, provides a novel antibiotic family, Albicidins, produced by three novel biosynthetic gene clusters (XALB1, XALB2, and XALB3) contained within a host cell DNA in which one strand comprises non-contiguously SEQ. ID No. 1, SEQ. ID No. 2 and SEQ ID No. 3, and the cell expresses the DNA to provide peptides including those named AlbI (SEQ ID No. 26) (encoded by SEQ ID No. 20), AlbII (SEQ ID No. 27) (encoded by SEQ ID No. 21), AlbIII (SEQ ID No. 28) (encoded by SEQ ID No. 22), AlbIV (SEQ ID No. 29) (encoded by SEQ ID No. 23), AlbVI (SEQ ID No. 31) (encoded by SEQ ID No. 18), AlbVII (SEQ ID No. 32) (encoded by SEQ ID No. 17), AlbVIII (SEQ ID No. 33) (encoded by SEQ ID No. 16), AlbIX (SEQ ID No. 34) (encoded by SEQ ID No. 15), AlbX (SEQ ID No. 35) (encoded by SEQ ID No. 10), AlbXI (SEQ ID No. 36) (encoded by SEQ ID No. 9), AlbXII (SEQ ID No. 37) (encoded by SEQ ID No. 8), AlbXIII (SEQ ID No. 38) (encoded by SEQ ID No. 7), AlbXIV (SEQ ID No. 39) (encoded by SEQ ID No. 6), AlbXV (SEQ ID No. 40) (encoded by SEQ ID No. 5), AlbXVII (SEQ ID No. 42) (encoded by SEQ ID No. 11), AlbXVIII (SEQ ID No. 43) (encoded by SEQ ID No. 12), AlbXIX (SEQ ID No. 44) (encoded by SEQ ID No. 13), AlbXX (SEQ ID No. 45) (encoded by SEQ ID No. 14), AlbXXI (SEQ ID No. 46) (encoded by SEQ ID No. 24), and AlbXXII (SEQ ID No. 47) (encoded by SEQ ID No. 25), that in turn interact within the host cell to produce one or more antibiotics as more fully illustrated in FIG. 11.
In one embodiment, the invention comprises a plurality of isolated and purified DNA strands which comprise nucleotide sequences selected from the group consisting of SEQ ID No: 1 to SEQ. ID No. 25, each individual sequence, except the transposases AlbV (SEQ ID No. 30) (encoded by SEQ ID No. 19) and AlbXVI (SEQ ID No. 41) (encoded by SEQ ID No. 4) found in the XALB1 cluster, being necessary to the biosynthesis of the novel family of antibiotics, Albicidins.
The invention also includes the peptides or proteins encoded by the genes of the biosynthetic complex expressed by the combination of DNA with a strand having sequences SEQ ID Nos. 1 to 3. Proteins are named with roman numerals and the prefix Alb from AlbI to Alb XXII have the amino acid sequences of SEQ ID Nos. 26 to 47 (not in Roman numeral order but in the order of placement of the genes within sequences SEQ ID Nos. 1 to 3 that express each protein). Expression of the peptides having the amino acid sequences of SEQ ID Nos. 26 to 29, 31 to 40 and 42 to 47, have been found to be all required for the successful biosynthesis of Albicidins.
The invention further provides a method for producing Albicidins comprising providing a modified host cell with a heterologous DNA Albicidin Biosynthetic Gene Cluster or set of genes defined as DNA operably comprising DNA sequences substantially similar to SEQ ID Nos. 1 to 3. Substantially the same means DNA having sufficient homology to provide expressed proteins that function to provide an antibiotic material having the structural components identified herein. Preferably a given sequence will have at least 70 percent homology to one of SEQ ID Nos. 1 to 3, preferably 85% homology and most preferably at least 95% homology. The method includes the steps of modifying the DNA of the host cell to comprise an operable expression system for maintaining the modified host cell under conditions supporting biosynthesis of Albicidins and isolation of Albicidins from the host cell or its environment. The invention further provides a method of production of a group of novel antibiotic materials utilizing at least three of the Sequences selected from the group consisting of DNA SEQ ID No. 1 to SEQ ID No. 25 (excluding transposases encoded by SEQ ID Nos. 4 and 19) inclusive in combination with additional sequences to produce a modified Albicidin-like material.
More specifically, the invention provides DNA Sequences comprising at least about 68,498 base pairs and including an about 55,839 bp region from the genome of X. albilineans designated as XALB1 (Albicidin Biosynthetic Gene Cluster 1; SEQ ID. No. 1) an additional non-contiguous region of about 2,986 bp, XALB2 (Albicidin Biosynthetic Gene Cluster 2; SEQ ID. No. 2), and a third region of about 9,673 bp, XALB3 (Albicidin Biosynthetic Gene Cluster 3; SEQ ID. No. 3). Albicidin Biosynthetic Gene Clusters 1-3 may be referred to, collectively, as the Albicidin Biosynthetic Gene Clusters and these sequences were found to be required for biosynthesis of Albicidins. Homology analysis revealed the presence of (i) four large genes with a modular architecture characteristic of polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) potentially involved in albicidin precursor biosynthesis; (ii) four smaller genes potentially involved in albicidin substrate biosynthesis (iii) four modifying genes; (iv) one enzyme activating gene, (v) two regulatory genes, (vi) one chaperone gene, (vii) two genes of unknown function; and (viii) two resistance genes. These are named and discussed more fully below. Together these genes allow the successful operation of the biosynthetic pathway when cloned into suitable host cells.
Alignment of individual NRPS and PKS domains revealed an extraordinary biosynthetic apparatus believed to involve a trans-action of separate PKS and NRPS domains which could contribute to the production of multiple, structurally related albicidins by the same gene cluster. Furthermore, analysis of selectivity-conferring residues indicated that four NRPS modules of XALB1 specify an unusual substrate.
In an alternate embodiment the invention provides a method of producing a polyketide carrying para-aminobenzoic acid and/or carbamoyl benzoic acid by inserting at least one DNA fragment that encodes a PKS protein into a cell and causing the cell to express the encoded PKS protein under conditions such that the PKS protein functions to produce a polyketide carrying either a para-aminobenzoic acid or a carbamoyl benzoic acid or both. Another embodiment provides a method of producing polyketide/peptides carrying para-aminobenzoic acid and/or carbamoyl benzoic acid by inserting at least one DNA fragment that encodes a PKS protein into a cell and causing the cell to express the encoded PKS protein under conditions such that the PKS protein functions to produce a polyketide carrying either a para-aminobenzoic acid or a carbamoyl benzoic acid or both. In yet another embodiment, the invention provides a method of activating nonproteinogenic amino acids like paraminobenzoic acid and/or carbamoyl benzoic acid for incorporation into peptides or polyketides by inserting at least one DNA fragment that encodes a PKS protein into a cell and causing the cell to express the encoded PKS protein under conditions such that the PKS protein functions to produce a polyketide carrying either a para-aminobenzoic acid or a carbamoyl benzoic acid or both.
There are three regions of the X. albilineans genome specifying albicidin production. XALB2 and XALB3 regions each contain only one gene, both of which are required for post-translational activation and folding of albicidin PKS and NRPS enzymes. The XALB1, XALB2 and XALB3 gene clusters are characterized by an unusual hybrid NRPS-PKS system, indicating that albicidin biosynthesis may provide an excellent model for investigating the biosynthesis of hybrid polyketide-polypeptide metabolites in bacteria. The availability of three genomic regions involved in albicidin production, XALB1 and XALB2 and XALB3, also offers the ability to express individually the enzymes of the albicidin family biosynthetic pathway including structural, resistance, secretory and regulatory elements, and to engineer overproduction of albicidin in mutated or modified host cells of the invention. The invention overcomes prior art limitations in albicidin production due to low yields of toxin production in X. albilineans and may also allow characterization of the chemical structure of albicidin as well as application of this potent inhibitor of prokaryote DNA replication.
The invention results from a number of unpredictable results namely the number and complexity of the enzymes involved in biosynthesis. The discovery of the complete sequence required for biosynthesis of Albicidins is previously unreported. The invention provides for a novel process for production of molecule having a polyketide-polypeptide backbone and the formula C40H35O15N6, a molecular weight of 839, and the structural elements shown in FIG. 11.
The invention further includes (a) the Albicidin Family Biosynthetic Gene Cluster including (b) the structural and regulatory elements of the operons that encode c) the enzymes PKS-1, PKS-2, PKS-3, PKS4, NRPS-1, NRPS-2, NRPS-3, NRPS-4, NRPS-5, NRPS-6 and NRPS-7 as well as (d) the proteins AlbI to AlbXXII, (e) the isolated enzymes, proteins, and active forms thereof, as well as mutants, fragments, and fusion proteins comprising any of the forgoing; (f) the uses of the enzymes or proteins encoded by the Albicidins Biosynthesis Gene Cluster or any one of its operons, (g) a host cell expressing one or more enzymes or proteins encoded by the Albicidin Family Biosynthetic Gene Cluster; (h) use of host cells having the Albicidins Biosynthesis Gene Cluster to produce an antibiotic; (i) methods of modifying the DNA sequences to produce members of a series of antibiotic compounds having structures related to Albicidins; (j) DNA sequences that encode the same proteins as any of SEQ. ID. Nos. 1 to 25 but differ in specific codons due to the multiplicity of codons that lead to expression of the same amino acid; (k) antibiotics produced by the process of expression of the Albicidin Family Biosynthetic Genes in a genetically modified host cell sustained in a culture medium and thereafter separation of the antibiotic from the host cell and culture medium; (l) an isolated and purified antibiotic produced by a process that includes at least three proteins coded by DNA sequences selected for the group consisting of SEQ. ID Nos. 1 to 25 in combination with additional enzymes that modify the product to provide a non-naturally occurring Albicidins like product having at least one of the useful properties reported for albicidin; and (m) a process for producing an antibiotic that comprises modifying a host cell to enhance expression of the DNA of the Albicidin Family Biosynthetic Gene Cluster by insertion of expression enhancing DNA into the genome of a Xanthomonas albilineans strain in a position operative to enhance expression of the enzymes of the Albicidin Family Biosynthetic Gene Cluster, culturing the modified host cell to produce an antibiotic and isolating the antibiotic. The products and methods described above have utility as proteins or as nucleic acids as the case may be, including such uses sources of pyrimidine or purine bases or amino acids, or as animal food supplements and the like, as well as the more important uses to provide antibiotics, plant disease treatment methods, genetically modified disease resistant plants, phytotoxins and the like.
The subject invention also provides an isolated and purified antibiotic produced by a process that includes at least three proteins coded by the nucleic acids of the subject invention in combination with additional enzymes that modify the product to provide a non-naturally occurring Albicidin-like product having at least one of the useful properties reported for albicidin. In certain embodiments, the antibiotic or antibiotics have at least one of the general structures illustrated in FIG. 11. In other embodiments, antibiotics of the subject invention have at least 4 of the structural elements illustrated in FIG. 11, and an elemental composition of C40H35N6O15.
The invention further provides a method of protecting a plant against damage from albicidin that comprises applying an agent that blocks expression at least one gene in the Albicidin Biosynthetic Gene Clusters to the plant to be protected. Additional inventions include a method of obtaining agents useful in blocking expression of albicidin by screening materials against a modified host cell line that expresses the Albicidin Biosynthesis Gene Clusters and selecting for materials that stop or decrease albicidin production and a method of protecting a plant; against phytotoxic damage from an antibiotic that comprises inserting into the plant and operably expressing at least one resistance gene from the Albicidin Biosynthesis Gene Clusters into the plant to be protected.
EXAMPLE 1
Materials and Methods
Bacterial strains and plasmids. The source of bacterial strains and their relevant characteristics are described in Table 1.
Media, antibiotics, and culture conditions. X. albilineans strains were routinely cultured on modified Wilbrink=s (MW) medium at 30C without benomyl (Rott et al., 1994). For long-term storage, highly turbid distilled water suspensions of X. albilineans were supplemented with glycerol to 15% (vol/vol) and frozen at B80C. For X. albilineans, MW medium was supplemented with the following antibiotics as required at the concentrations indicated: kanamnycin, 10 or 25 μg/ml; and rifampicin, 50 μg/ml. E. coli strains were grown on Luria-Bertani (LB) agar or in LB broth at 37C and were maintained and stored according to standard protocols (Sambrook et al., 1989). For E. coli, LB medium was supplemented with the following antibiotics as required at the concentrations indicated: kanamycin, 50 μg/ml; ampicillin, 50 μg/ml.
Bacterial conjugation. DNA transfer between E. coli donor (DH5_MCR/pAlb389 or pAC389.1, Table 1) and rifampicin-resistant X. albilineans recipients (X. strains AM10, AM12, AM13, AM36 and AM37, Table 1) was accomplished by triparental conjugation with plasmid pRK2073 as the helper as described previously (Rott et al., 1996).
Assay of albicidin production. Albicidin production was tested by a microbiological assay as described previously (Rott et al., 1996). Rifampicin and kanamycin exconjugants were spotted with sterile toothpicks (2-mm-diameter spots) onto plates of SPA medium (2% sucrose, 0.5% peptone, 1.5% agar) and incubated at 28° C. for 2-5 days. The plates were then overlaid with a mixture of E. coli DH5α (107 cells in 2 ml of distilled water) plus 2 ml of molten 1.5% (wt/vol) Noble agar (Difco) at ca. 65° C. and examined for inhibition zones after 24 h at 37 C.
Nucleic acid manipulations. Standard molecular techniques were used to manipulate DNA (Sambrook et al., 1989) except for total genomic DNA preparation. Total genomic DNA for southern blot hybridization was prepared as described by Gabriel and De Feyter (1992).
PCR Conditions. PCR amplifications were performed in an automated thermal cycler PTC-100TM (MJ Research, Inc). The 25 μl PCR reaction mix consisted of 100 ng of genomic DNA or 1 ng of plasmid DNA, 2.5 μl of 10×PCR buffer without MgCl2 (Eurobio), 80 μM dNTP mix, 2.5 units of EUROBIOTAQII® (Eurobio), 25 pmoles of each primer, 2.0 mM MgCl2 (Eurobio) and sterilized distilled water to final volume. The PCR program was 95° C. for 2 min, 25 cycles at 94° C. for 1 min, Tm for 1 min and 72° C. for 1 min, with a final 72° C. extension for 5 min. Tm temperature was determined for each couple of primers and varied between 55° C. and 60° C. A 5 μl aliquot of each amplified product was analyzed by electrophoresis through a 1% agarose gel. For sequencing, PCR products were cloned with the pGEM®-T Easy Vector System (Promega).
Oligonucleotide synthesis. Oligonucleotides were purchased from Genome Express (Grenoble or Montreuil, France).
DNA sequencing. Automated DNA sequencing was carried out on double-stranded DNA by the dideoxynucleotide chain termination (Sanger et al., 1977) using a Dye Terminator Cycle Sequencing kit and an ABI Perkin-Elmer sequencer according to the manufacturer's procedure. Both DNA strands were sequenced with universal primers or with internal primers (20mers). This service was provided by Genome Express (Grenoble, France). Computer-aided sequence analyses were carried out using Sequence NavigatorTM (Applied Biosystems, Inc) and SeqMan (DNASTAR Inc.) programs.
Sequence analysis. Nucleotide sequences were translated in all six reading frames using EditSeq (DNASTAR Inc.). Potential products of ORFs longer than 100 b were compared to protein databases by the PSI-BLAST program (Swiss-Prot and Genbank) on the NCBI with site (ncbi.nlm.nih.gov/) using Altschul program (Altschul et al., 1997). The TERMINATOR program of the Genetics Computer Group was used to identify putative Rho-independent transcription terminators.
Procedures
EXAMPLE 2
Sequencing of the Double Strand Region of 55,839 Bp from X. albilineans Containing XALB1 SEQ ID NO. 1
In FIG. 1 is presented a physical map and genetic organization of XALB1. In the figure, E and K are restriction endonuclease sites for EcoRI and KpnI respectively. Rectangular boxes represent DNA fragments labeled A through N. The numbers below each rectangular box are the number of Tn5-gus insertion sites previously located in each DNA fragment (Rott et al., 1996). The DNA inserts carried by plasmids pALB571 and pALB540 are represented by bold bars above the physical map. The location and direction of putative orfs identified in the XALB1 gene cluster are shown by arrows. Precise positions and proposed functions for individual orfs are summarized in Tables 2 and 3, respectively. Position of insertional sites of eight albicidin-defective mutants determined by sequencing are indicated by vertical arrows. The location and direction of putative ORFS identified in the XALB1 gene cluster are shown by arrow shapes. These twenty putative ORFs are potentially organized in four or five operons, as indicated at the bottom of the figure. Patterns indicate NRPS and PKS genes (diagonal crosshatch), methyl transferase and esterase genes (hollow rectangles), carbamoyl transferase gene (fine crosshatch), benzoate-derived products biosynthesis genes (white), regulatory genes (vertical lined), resistance genes (diagonal lines) and other genes with function of unknown significance to albicidin production (black), and three insertional sites of eight albicidin-defective mutants determinated by sequencing are indicated by vertical arrows. Dotted regions in the physical map and in ORFs represent the two internal duplicated DNA regions of XALB1.
The sequence illustrated in FIG. 1 was generated as follows. The sources of DNA are set out in Table 1. DNA fragments F, E, B, C, I, and G, generated by the digestion of cosmid pALB571 (Rott et al., 1996) with EcoRI and/or KpnI, were subcloned into pBCKS (+) and were sequenced from the resulting subclones, pBC/F, pBC/E, pBC/B, pBC/C, pBC/I and pBC/G. DNA fragment D′ which corresponds to the part of fragment D present in cosmid pALB571 was sequenced from plasmid pUFR043/D′ obtained following self ligation of the complete EcoRI digested cosmid pALB571. DNA fragment H was sequenced from pAM45.1 (Rott et al., 1996), obtained following cloning into vector pBR325 of the 12 kb EcoRI fragment carrying Tn5 and flanking sequences from mutant strain XaAM45. DNA fragment A′ contains the part of fragment A present in cosmid pALB571 and was subcloned into vector pBCKS (+) and the resulting plasmid pBC/A′ was used for sequencing. The presence of a large internal duplication made alignment of sequence data obtained from pBC/A′ difficult. This difficulty was resolved using sequence data obtained from an additional plasmid, pAM4, obtained following cloning into vector pBluescript II KS (+) of the 12 kb EcoRI fragment carrying Tn5 and flanking sequences from mutant strain XaAM4, which contains only one copy of the large internal duplication. Sequence data from pBC/A′ were used to determine the first 1542 bp of fragment A′ between nucleotides C-19001 and G-20543. Sequence data from pAM4 and pBC/A′ were used to determine the last 4823 bp of fragment A′ between nucleotides G-21653 and G-26477. The overlapping region between nucleotides G-20469 and C-22159 was amplified by PCR from cosmid pALB571 using primers contig13-1160 (5′gcgtaccgttgtccagtagg3′) SEQ ID NO. 48 and pAM4-14 (5′gctggaaaccgagaatctga3′) SEQ ID NO. 49, and was sequenced. Resulting sequence data were used to complete sequencing of DNA fragment A′. The junctions A/F, F/H, H/E, E/B, B/C, C/I, I/G, G/D between corresponding DNA fragments were sequenced directly from cosmid pALB571. EcoRI DNA fragment containing fragments A and F was subcloned from pALB540 into pBCKS (+), and the resulting plasmid pBC/AF was used to determine the part of DNA fragment A which was not present in cosmid pALB571 between nucleotides G-13682 and G-19001. EcoRI DNA fragments J, K, L, N were subcloned from pALB540 into pBCKS (+) and were sequenced from resulting plasmid pBC/J, pBC/K, pBC/L, and pBC/N. The junctions L/K, K/J and J/A between corresponding DNA fragments were sequenced directly from cosmid pALB540. DNA region between nucleotides G-7517 and T-8721 was amplified by PCR from cosmid pALB540 using primers E114 (5′gacacgatcagccgctagga3′) SEQ ID NO. 50 and EI4-380 (5′accagcagttgggccagcct3′) SEQ ID NO. 51 and was sequenced. Resulting sequence data were used to determine the sequence of fragment M and of junctions N/M and M/L. The nucleotide sequence of 55,839 bp containing the entire major gene cluster involved in Albicidin production was sequenced on both strands.
EXAMPLE 3
Analysis of the Large Internal Duplications in the DNA Sequence of XALB1
The sequence of the 55,839 bp genomic region (SEQ ID NO. 1) contains two large internal duplications as shown by the dotted regions in the physical map of FIG. 1. A direct duplication of 1736 bp was located in DNA fragment A between nucleotides G-19904 and G-21639 and between nucleotides G-23057 and G-24792. Another direct duplication of a 2727 bp was found in DNA fragments B and C between nucleotides C-40410 and G-43136 and between nucleotides C-46644 and G-49370. Comparison of the two copies of each duplication revealed that the two copies of the 1736 bp duplication are identical except for one nucleotide at position 21058, and that the two copies of the 2727 bp duplication are 98.8% identical and differ by 30 nucleotides.
EXAMPLE 4
Comparison of XALB1 with the xabB EcoRI Fragment
Comparison of the DNA sequence of the 55,839 bp genomic region described in this study with the partial DNA sequence of 16,511 bp of the same region in Huang et al., 2001 (described by Huang et al. as an EcoRI fragment including full length xabB from X. albilineans strain Xa13 [GenBank accession No. AF239749]), revealed that the DNA sequence from strain Xa13 over 16,511 bp is identical to the sequence from strain Xa23R1, described herein, with the following exceptions: 1) five nucleotides are different at positions 42963, 42972, 42980, 43014 and 43071 of the XALB1 sequence, and 2) nucleotides from positions 43137 to 49370 are missing (internal to albI; refer FIG. 1). Analysis of genomic DNA of seven strains isolated from different countries (Australia, Reunion Island, Kenya, Zimbabwe and USA), digested by KpnI and hybridized with the pBC/C plasmid (Table 1) labeled with 32P, revealed that two DNA fragments corresponding to the XALB1 fragments B and C were present in all strains (data not shown). This result indicated that all studied strains contain albI and not xabB because in albI the pBC/C plasmid probe hybridizes with the large internal duplication present in both DNA fragments B and C (FIG. 1). Based on this observation we postulated that the DNA sequence of XabB reported as full length by Birch in PCT WO 02/24736 A1 (Their seq. ID#1) appears to be incomplete and missing 6,234 bp of DNA sequence encoding 2,078 amino acids.
EXAMPLE 5
Reading Frame Analysis in XALB1
Analysis of the 55,839 bp double strand region for coding sequences revealed the presence of 20 open reading frames (ORFs) designated albI to albXX (Table 2 below) which are distributed in four groups of genes according to their position and their orientation in the XALB1 cluster (FIG. 1). Genes of each group may form part of the same operon as judged by their overlapping stop and start codons, or by the relatively short intergenic region which varies from 5 to 274 nucleotides. The 20 ORFs appear to be organized in four operons: operon 1 formed by albI-albIV; operon 2 by albV-albIX; operon 3 by albX-albXVI; operon 4 by albXVII-albXX. The majority of alb ORFs are initiated with an ATG codon, except albI and albXVII which are initiated with a TTG codon, and albIV and albVI which are initiated with a GTG start codon. In seven ORFs of XALB1, start codons are preceded by the consensus sequence GAGG which may correspond to the ribosome binding site. Other ORFs are preceded by a less conserved sequence which contain at least three nucleotides A or G and which may serve as a weak ribosome binding site.
EXAMPLE 6
Sequencing of the Tn5 Insertional Site of Eight Tox− Mutants Previously Located in XALB1
Eight of the 45 X. albilineans Tox− mutants complemented by cosmid pALB540 and/or cosmid pALB571 and previously described (Rott et al., 1996) were further analyzed. All eight mutants contain a single Tn5 insertion and correspond to the following X. albilineans strains: XaAM7, XaAM15, XaAM45, and XaAM52 which are complemented by pALB571 but not by pALB540; XaAM4, XaAM29 and XaAM40 which are complemented by both cosmids; and XaAM1 which is complemented by pALB540 but not by pALB571. The Tn5 insertional site of each Tox− mutant was sequenced from plasmids obtained following cloning in pBR325 or in pBluescript II KS (+) of the EcoRI fragments carrying Tn5 and flanking sequence using the sequencing primer GUSN (5′tgeccacaggccgtcgagt3′) SEQ ID No. 52 that annealed 135 bp downstream from the insertional sequence IS50L of Tn5-gusA. The sequence of the Tn5 insertional site was compared with the 55,839 bp sequence containing XALB1 in order to determine the alb gene disrupted in each Tox− mutant. albI is disrupted by the Tn5 insertion in XaAM15 and XaAM45 at position 33443 and 34229, respectively (FIG. 1). albIV is disrupted by the Tn5 insertion in XaAM7 and XaAM52 at position 53704 and 53915, respectively. albIX is disrupted by the Tn5 insertion in XaAM4, XaAM29 and XaAM40 at position 21653, 23444 and 24376, respectively. alb XI is disrupted by the Tn5 insertion in XaAM1 at position 13301. These results are in accordance with the previous characterization of Tox− mutants using Southern blot hybridization Rott et al., 1996), except for XaAM1. The Tn5-gusA insertion site of XaAM1 was previously located in DNA fragment A (Rott et al., 1996) but results of this study showed that this site is located in DNA fragment J (FIG. 1).
EXAMPLE 7
Homology Analysis of Proteins Potentially Encoded by XALB1
Preliminary functional assignments of individual ORFs were made by comparison of the deduced gene products with proteins of known functions in the Genbank database. The results are set out in Table 3 below. Among the ORFs identified from the sequenced XALB1 gene cluster, we found (i) four genes, albI SEQ ID No. 20, albIV SEQ ID No. 23, albVII SEQ ID No. 17 and albIX SEQ ID No. 15, encoding PKS and/or NRPS modules; (ii) one carbamoyl transferase gene, albXV SEQ ID No. 5; (iii) two esterase genes, albXI SEQ ID No. 9 and albXIII SEQ ID No. 7; (iv) two methyltransferase genes, albII SEQ ID No. 21 and albVI SEQ ID No. 18; (v) two benzoate-derived products biosynthesis genes, albXVII SEQ ID No. 11 and albXX SEQ ID No. 14; (vi) two putative albicidin biosynthesis regulatory genes, albIII SEQ ID No. 22 and alb VIII SEQ ID No. 16; (vii) two putative albicidin resistance genes, albXIV SEQ D No. 6 and albXIX SEQ ID No. 13; and (viii) two additional ORFs encoding proteins similar to transposition proteins, albV SEQ ID No. 19 and albXVI SEQ ID No. 4. No known function was found in the database for albX SEQ ID No. 10 and albXII SEQ ID No. 8. The potential product of albXVIII SEQ ID No. 12 appeared to be a truncation of an enzyme with strong similarity to 4-amino-4-deoxychorismate lyase and branched-chain amino acid aminotransferase. Since the gene encoding the predicted product is roughly half the length of other such lyase or aminotransferase genes, albXVIII may be the result of a recombination event and may be non functional.
EXAMPLE 8
The alb PKS and/or NRPS Genes
The potential product of albI, designated AlbI SEQ ID No. 20, is a protein of 6879 aa with a predicted size of 755.9 kDa. This protein is very similar to the potential product of the xabB gene from X. albilineans strain Xa13 from Australia (Huang et al., 2001), but it differs in length and size (See Table 4 below). XabB is a protein of 4801 amino acids with a predicted size of 525.7 kDa. Comparison of AlbI with XabB revealed that the N-terminal regions from Met-1 to Ile-4325 of both proteins are identical except for five amino-acids which are Tyr-3941, Pro-3952, Ala-4054, Ala-4271 and Gln-4284 in AlbI and His-3941, Ala-3952, Val-4054, Val-4271 and Glu-4284 in XabB. The same comparison revealed that the AlbI C-terminal region from Arg-6404 to the stop codon is 100% identical to the XabB C-terminal region from Arg-4326 to the stop codon.
The N-terminal region (from Met-1 to Asp-3235) of AlbI is 100% identical to the corresponding region in XabB which was previously described as similar to many microbial modular PKS (Huang et al., 2001). This PKS region may be divided into three modules (FIG. 2). Abbreviations used in the Figure are: A, adenylation; ACP, acyl carrier protein; AL, acyl-CoA ligase; C, condensation; KR, β-ketoacyl reductase; KS, β-ketoacyl synthase; NRPS, nonribosomal peptide synthase; PCP, peptidyl carrier protein; PKS, polyketide synthase; TE, thioesterase; HBCL, 4-hydroxybenzoate-CoA ligase. The question mark in the NRPS-2 domain indicates that this A domain is incomplete. The first module designated PKS-1 contains acyl-CoA ligase (AL) and acyl carrier protein (ACP1) domains. The second module designated PKS-2 contains J-ketoacyl synthase (KS1) and β-ketoacyl reductase (KR) domains followed by two consecutive ACP domains (ACP2 and ACP3). The third module designated PKS-3 contains a KS domain (KS2) followed by a PCP domain (PCP1). Apart from their very high similarity with XabB, these three PKS modules exhibited the highest degree of overall similarity with polyketide synthases SafB and PksM from Myxococcus xanthus and Bacillus subtilis, respectively (Table 4). The motifs characteristic of these domains are 100% identical to those of XabB which were previously aligned with those from other organisms (Huang et al., 2001). The AL domain contains the conserved adenylation core sequence (SGSSG) and the ATPase motif (TGD). The three ACP domains contain a 4′-phosphopantetheinyl-binding cofactor box GxDS(IL), except that A replaced G in ACP1. Both KS domains contain motif GPxxxxxxxCSxSL around the active site Cys, and two His residues downstream from the active site Cys, in motifs characteristic of these enzymes. The KR domain contains the NAD(P)H-binding site GGxGxLG.
The PKS part of AlbI is linked by the PCP1 domain to the four apparent nonribosomal peptide synthase modules designated NRPS-1, NRPS-2, NRPS-3 and NRPS-4 (FIG. 2). NRPS-1, NRPS-2 and NRPS-3 modules display the ordered condensation, adenylation (A) and PCP domains typical of such enzymes (Marahiel et al., 1997), and NRPS-4 consists of an extra C domain which may correspond to an incomplete NRPS module. Known conserved sequences, characteristic of the domains commonly found in peptide synthases (Marahiel et al., 1997), were compared to those from NRPS-1, NRPS-2, NRPS-3 and NRPS-4 (Tables 5, 6 and 7). Sequences characteristic of C, A, or PCP domains are conserved in these four NRPS, except in A domain of NRPS-2 module, suggesting that this latter A domain may be not functional. Comparison of the four NRPS modules among themselves revealed that NRPS-2, NRPS-3 and NRPS-4 modules were 30.7%, 94.4% and 47.5% similar to NRPS-1 module, respectively. Comparison with XabB revealed NRPS-2 and NRPS-3 modules were not present in XabB which contains only NRPS-1 and NRPS-4 modules (FIG. 2). The dotted box in FIG. 2 corresponds to the apparent deletion of the NRPS-2 and NRPS-3 modules in XabB as compared to AlbI. Apart their very high similarity with XabB, Alb I NRPS modules exhibited the highest degree of overall similarity with non-ribosomal peptide synthases NosA and NosC from Nostoc sp.
albIV potentially encodes a protein of 941 aa (AlbIV) with a predicted size of 104.8 kDa. AlbIV is similar to several non-ribosomal peptide synthases such as the BA3 peptide synthase involved in bacitracin biosynthesis in Bacillus licheniformis (Table 4). AlbIV forms one NRPS module designated NRPS-5 that contains only an A domain and a PCP domain (FIG. 2). Sequences characteristic of the domains A and PCP commonly found in peptide synthases (Marahiel et al., 1997) are conserved in AlbIV (Tables 6 and 7). However the A domain present in AlbIV differs from A domains commonly found in peptide synthases: conserved sequences corresponding to cores A8 and A9 in AlbIV are separated by a very long peptide sequence of 390 amino-acids. This additional peptide sequence exhibits a significative similarity with the protein WbpG of 377 amino acids involved in the biosynthesis of a lipopolysaccharide in Pseudomonas aeruginosa (Table 4).
albVII potentially encodes a protein of 765 aa (AlbVII) with a predicted size of 83.0 kDa similar to the 4-hydroxybenzoate-CoA ligase from several bacteria and the closest protein (HbaA) was from Rhodopseudomonas palustris (Table 4). High similarity between AlbVII and HbaA suggests that AlbVII is a 4-hydroxybenzoate-CoA ligase and constitutes a fourth PKS module designed PKS-4. The size of HbaA is smaller (539 aa) and the similarity between the two proteins starts only at the residue 277 of AlbVII and at the residue 28 of HbaA. Comparison of AlbVII sequence located upstream from residue 277 produced no significant alignment AlbVII, like 4-hydroxybenzoate-CoA ligases, contains some conserved sequences characteristic of the A domain commonly found in peptide synthases (Table 6).
albIX encodes a protein of 1959 aa (AlbIX) with a predicted size of 218.4 kDa similar to non-ribosomal peptide synthases. Known conserved sequences, characteristic of the domains commonly found in peptide synthases (Marahiel et al., 1997), were compared with those from AlbIX which forms two NRPS modules designated NRPS-6 and NRPS-7 (Tables 5, 6 and 7). NRPS-6 contains only one A and one PCP domain. NRPS-7 contains the three domains characteristic of NRPS modules (A-C-PCP) followed by a TE domain (FIG. 2). Apart their very high similarity with XabB, NRPS-6 and NRPS-7 modules exhibited the highest degree of overall similarity and identity with non-ribosomal peptide synthases DhbF from B. subtilis and NosA from Nostoc sp. (Table 4).
EXAMPLE 9
The alb Carbamoyl Transferase Gene
albXV potentially encodes a protein of 584 aa with a predicted size of 65.2 kDa. This protein, AlbXV, is similar to BlmD, a carbamoyl transferase involved in bleomycin biosynthesis in Streptomyces vertillus (Du et al., 2000), and to a probable carbamoyl transferase potentially expressed in P. aeruginosa (Table 4). High similarity of AlbXV with these proteins suggests that AlbXV is a carbamoyl transferase.
EXAMPLE 10
The alb Esterase Genes
albXV potentially encodes a protein of 315 aa with a predicted size of 35.9 kDa. This protein, AlbXI, exhibits low similarity to SyrC, a putative thioesterase involved in syringomycin biosynthesis by Pseudomonas syringae (Zhang et al., 1995), and to a potential hydrolase encoded by Streptomyces coelicolor (Table 4). Precise function of SyrC remains unknown but SyrC is similar to a number of thioesterases, including fatty acid thioesterases, haloperoxidases, and acyltransferases that contain a characteristic GxCxG motif. The corresponding SyrC domain GICAG is conserved in AlbXI which contains the sequence GWCQA, except that A replaces the last G, suggesting that AlbXI may be an esterase despite its low overall similarity with SyrC.
albXIII potentially encodes a protein of 317 aa with a predicted size of 34.5 kDa. This protein, AlbXIII, is similar to hypothetical proteins with unknown function from several bacteria including Caulobacter crescentus (Table 4). AlbXIII and these hypothetical proteins contain a GxSxG motif characteristic of serine esterases and thioesterases, the corresponding sequence in AlbXIII being GHSVG. In addition, AlbXIII presents a similarity with the 2-acetyl-1-alkylglycerophosphocholine esterase which hydrolyzes the platelet-activating factor in Canis familiaris (Table 4), suggesting that AlbXIII is an esterase.
EXAMPLE 11
The alb Methyltransferase Genes
albII potentially encodes a protein of 343 aa (AlbII) with a predicted size of 37.7 kDa. albII is 100% identical to the xabC cistron, previously described as encoding an O-methyltransferase downstream xabB (Huang et al., 2000a). This conclusion is based on the similarity of XabC with a family of methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a co-substrate for O-methylation including TcmO protein from Streptomyces glaucescens (Huang et al., 2000a). AlbII contains three highly conserved motifs of SAM-dependent methyltransferases, including the motifI involved in SAM binding (FIG. 3). In the Figure, identical or similar amino acids (A=G; D=E; I=L=V) are shown in bold. Numbers indicate the position of the amino acid from the N-terminus of the protein. Abbreviations used in the Figure are: Sgl-TcmO (SEQ ID NOs: 55, 56 and 57) and Sgl-TcmN (SEQ ID NOs: 58, 59 and 60), multifunctional cyclase-hydratase-3-O-Mtase and tetracenomycin polyketide synthesis 8-O-Mtase of Streptomyces glaucescens, respectively (accession number: M80674); Smy-MdmC, midecamycin-O-Mtase of Streptomyces mycarofaciens (accession number: M93958) (SEQ ID NOs: 61, 62 and 63); Mxa-SafC, Saframycin O-Mtase of Myxococcus xanthus (accession number: U24657) (SEQ ID NO:s: 64, 65 and 66); Ser-EryG, erythromycin biosynthesis O-Mtase of Saccharopolyspora erythraea (accession number: S18533) (SEQ ID NOs: 67, 68 and 69); Spe-DauK, carminomycin 4-O-Mtase of Streptomyces peucetius (accession number: L13453) (SEQ ID NOs: 70, 71 and 72); Sal-DmpM, O-demethylpuromycin-O-Mtase of Streptomyces alboniger (accession number: M74560) (SEQ ID NOs: 73, 74 and 75); Shy-RapM, rapamycin O-Mtase of Streptomyces hygroscopicus (accession number: X86780) (SEQ ID NOs: 76, 77 and 78) ; Sav-AveD, avermectin B 5-O-Mtase of Streptomyces avermitilis (accession number: G5921167) (SEQ ID NOs: 79, 80 and 81), Sar-Cmet, mithramycin C-methyltransferase of Streptomyces argillaceus (accession number: AF077869) SEQ ID NOs: 82, 83 and 84); AlbII, putative albicidin biosynthesis C-Methyltransferase of Xanthomonas albilineans (SEQ ID No. 27); identical to XabC, accession number: AF239749) (SEQ ID NOs: 85, 86 and 87).
Comparison of AlbII with the Genbank database revealed that AlbII, besides 100% identity to XabC, exhibited the highest degree of overall identity with MtmMII, a C-methyltransferase from Streptomyces argillaceus (Table 4) involved in C-methylation of the polyketide chain for mithramycin biosynthesis, suggesting that AlbII is a C-methyltransferase. XabC was not compared by Birch and co-workers with MtmMII (Huang et al., 2000a) because the MtmMII sequence was not available until recently in the Genbank database. The three highly conserved motifs in SAM methyltransfererases are also present in MtmMII (FIG. 3), suggesting that AlbII is a C-methyltransferase SAM-dependent.
albVI potentially encodes a protein of 286 aa (AlbVI) with a predicted size of 32.1 kDa similar to several hypothetical protein from Mycobacterium tuberculosis (Genbank accessions No. AAK46042, AAK48238, AAK44517, AAK46218) and from S. coelicolor (Genbank accession No. CAC03631). AlbVI is also similar to the tetracenomycine C synthesis protein (TcmP) of Pasteurella multocida (Table 4). Four highly conserved motifs in TcmP and other O-methyltransferases are also present in AlbVI (FIG. 4), suggesting that AlbVI is an O-methyltransferase. In the Figure, identical or similar aa (A=G; D=E; I=L=V; K=R) are shown in bold. Numbers indicate the position of aa from the N-terminus of the protein. Abbreviations used in the Figure are: Sgl-tcmP, tetracenomycin C synthesis protein of Streptomyces glaucescens (accession number: C47127) (SEQ ID NOs: 88, 89, 90 and 91); Sme-PKS, putative polyketide synthase of Sinorhizobium meliloti (accession number: AAK65734) (SEQ ID NOs: 92, 93, 94 and 95); Pmu-tcmP: tetracenomycin C synthesis protein of Pasteurella multocida (accession number: AAK03406) (SEQ ID NOs: 96, 97, 98 and 99); Mtu-Omt: putative O-methyltransferase of Mycobacterium tuberculosis (accession number: AAK45444) (SEQ ID NOs: 100, 101, 102 and 103); Mlo-Hp: hypothetical protein containing similarity to O-methyltransferase of Mesorhizobium loti (accession number: BAB50127) (SEQ ID NOs: 104, 105, 106 and 107); Mtu-Hp: hypothetical protein of Mycobacterium tuberculosis (accession number: AAK46042) (SEQ ID NOs: 108, 109, 110 and 111); Mtu-Hp2: hypothetical protein of Mycobacterium tuberculosis (accession number: AAK48238) (SEQ ID NOs: 112, 113, 114 and 115); Mtu-Hp3: hypothetical protein of Mycobacterium tuberculosis (accession number: AAK44517)(SEQ ID NOs: 116, 117, 118 and 119); Mtu-Hp4: hypothetical protein of Mycobacterium tuberculosis (accession number: AAK46218) (SEQ ID NOs: 120, 121, 122 and 123); Sco-Hp: hypothetical protein of Streptomyces coelicolor (accession number: CAC03631) (SEQ ID NOs: 124, 125, 126 and 127); AlbVI, putative albicidin biosynthesis O-Methyltransferase of Xanthomonas albilineans (this study) (SEQ ID NOs: 128, 129, 130 and 131).
EXAMPLE 12
The alb Derived-benzoate Products Biosynthesis Genes
albXVII potentially encodes a protein of 716 aa with a predicted size of 79.8 kDa. This protein, AlbXVI, is very similar to the para-aminobenzoate (PABA) synthase from Streptomyces griseus (Table 4). This enzyme is required for the production of the antibiotic candicidin (Criado et al., 1993).
albXVII potentially encodes a protein of 137 aa with a predicted size of 15.0 kDa. This protein, AlbXIII, is similar to the 4-amino-4-deoxychorismate lyase (ADCL) from P. aeruginosa (Table 4). The function of ADCL is to convert 4-amino-4-deoxychorismate into PABA and pyruvate. The length of AlbXVIII is smaller (Table 4) than the length of ADCL and the similarity of AlbXVIII with this protein starts only at residue 161. albXVIII is preceded by a small ORF encoding a sequence of 59 amino acids similar to the first 42 amino acids of ADCL from P. aeruginosa. These data suggest that albXVIII is probably a truncated form of albXVIII and probably not functional. albXVIII may, therefore, not be involved in albicidin biosynthesis. The region between albXVII and albXVIII was amplified by PCR from total DNA of X. albilineans Xa23R1 strain using primers ORFW (5′gcgagaggacaagctgctgc3′) SEQ ID No. 53 and ORFY (5′cgttgaggatgcagcgctcg31′) SEQ ID No. 54 and was sequenced. Resulting sequence data showed that the sequence of the PCR fragment was 100% identical to the sequence of pALB540, indicating that the recombination of albXVIII did not occur during cloning of the genomic fragment in pALB540.
albXX potentially encodes a protein of 202 aa with a predicted size of 22.6 kDa. This protein AlbXX is similar to the 4-hydroxybenzoate synthase potentially involved in ubiquinone biosynthesis by Escherichia coli (Siebert et al., 1992).
EXAMPLE 13
The alb Regulatory Genes
albIII potentially encodes a protein of 167 amino acids with a predicted size of 17.8 kDa that is similar to the transcription factors ComA of different bacteria such as E. coli and B. licheniformis (Table 4). ComA transcription factors appear to be involved in regulation of antibiotic production in bacteria. In E. coli, a gene similar to comA is present in the enterobactin biosynthesis gene cluster (Liu et al., 1989). In B. subtilis, ComAB was described as a probable positive activator of lichenysin synthetase transcription (Yakimov et al., 1998) and a gene similar to comA was shown to be essential for bacilysin biosynthesis (Yazgan et al., 2001). These data suggest that AlbIII regulates transcription of genes involved in albicidin biosynthesis.
albVIII potentially encodes a protein of 330 aa with a predicted size of 37.7 kDa. This protein, AlbVIII, is very similar to the SyrP like protein from S. verticillus and to SyrP protein from P. syringae (Table 4). SyrP participates in a phosphorylation cascade controlling syringomycin synthesis (Zhang et al., 1997) and the syrP like gene was described in the S. verticillus bleomycin biosynthetic gene cluster (Du et al., 2000). These data suggest that AlbVIII regulates albicidin biosynthesis in X. albilineans. 
EXAMPLE 14
The alb Resistance Genes
albXIV potentially encodes a protein of 496 aa with a predicted size of 52.7 kDa. This protein, AlbXIV, is 100% identical to AlbF isolated from X. albilineans strain Xa13 (GenBank Accession AF403709; direct submission by Bostock and Birch and described as “a putative albicidin efflux pump which confers resistance to albicidin in E. coli”). AlbXIV and AlbF are closely related to a family of transmembane transporters involved in antibiotic export and antibiotic resistance in many antibiotic-producing organisms. AlbXIV and AlbF exhibited the highest degree of overall identity with the putative transmembrane efflux protein from S. coelicolor (Table 4). These data suggest that AlbXIV and AlbF may be involved in albicidin resistance by transporting the toxin out of the bacterial cells that produce it. Alternatively, AlbXIV and AlbF may simply play a role in antibiotic secretion and/or plant pathogenesis to effect the transport of albicidin outside of producing cells.
albXIX potentially encodes a protein of 200 aa with a predicted size of 22.8 kDa. This protein, AlbXIX, is similar to the McbG protein from E. coli (Table 4). In Enterobacteriae, the McbG protein, together with two other proteins (McbE and McbF), was shown to cause immunity to the peptide antibiotic microcin B17 which inhibits DNA replication by induction of the SOS repair system (Garrido et al., 1988). McbE and McbF proteins serve as a pump for the export of the active antibiotic from the cytoplasm, whereas a McbG alone also provides some protection: a well-characterized deficient-immunity phenotype is exhibited by microcin B17-producing cells in the absence of the immunity gene mcbG (Garrido et al., 1988). The significant similarity between AlbXIX and McbG, together with the fact that albicidin also blocks DNA replication (Birch and Patil, 1985a) suggests that AlbXIX confers immunity against albicidin in X. albilineans. 
EXAMPLE 15
Transposition Proteins
albV is 100% identical to the thp gene described in a divergent position upstream from xabB (Huang et al., 2000a). The thp gene potentially encodes a protein of 239 aa displaying significant similarity to the IS21-like transposition helper proteins. In X. albilineans strain LS155 from Australia, insertional mutagenesis of thp blocked albicidin production, but trans-complementation failed, indicating the involvement in albicidin production of a downstream gene in the thp operon (Huang et al., 2000a).
albXVI potentially encodes a protein of 88 aa with a predicted size of 9.8 kDa similar to the transposases from several bacteria such as Xanthomonas axonopodis or Desulfovibrio vulgaris (Table 4).
The presence of transposition proteins in the XALB1 cluster is probably a remnant from a past transposition event that may have contributed to the development of the albicidin XALB1 cluster.
EXAMPLE 16
Unknown Functions
AlbX potentially encodes a protein of 83 aa with a predicted size of 9.4 kDa. This protein, AlbX, is similar to an hypothetical protein from P. aeruginosa and to the MbtH protein from Mycobacterium tuberculosis. MbtH is a protein with unknown function found in the mycobactin gene cluster (Quadri et al., 1998). A MbtH-like protein with unknown function was also described in the bleomycin biosynthetic gene cluster of S. verticillus (Du et al., 2000). These data suggest that AlbX is involved in albicidin biosynthesis but its function remains unknown.
albXII potentially encodes a protein of 451 aa with a predicted size of 51.6 kDa. This protein, AlbXII, is very similar to a protein of 55 kDa encoded by the boxB gene in Azoarcus evansii (Table 4). This protein is a component of a multicomponent enzyme system involved in the hydroxylation of benzoyl CoA, a step of aerobic benzoate metabolism in Azoarcus evansii, but its function remains unknown (Mohamed et al., 2001).
EXAMPLE 17
Prediction of Amino Acid Specificity of Alb NRPS Modules
In NRPSs, specificity is mainly controlled by A domains which select and load a particular amino-, hydroxy- or carboxy-acid unit (Marahiel et al., 1997). The substrate-binding pocket of the phenylalanine adenylation (A) domain of the gramicidin S synthetase (GrsA) from Brevibacillus brevis was recently identified by crystal structure analysis as a stretch of about 100 amino acid residues between highly conserved motifs A4 and A5 (Conti et al., 1997). Based on sequence analysis of known A domains, in relation to the crystal structure of the GrsA (Phe) substrate binding pocket, similar models have been published to predict the amino acid substrate which is recognized by an unknown NRPS A domain (Challis et al., 2000; Stachelhaus et al., 1999). These models postulate specificity-conferring codes for A domains of NRPS consisting of critical amino acid residues putatively involved in substrate specificity. The model proposed by Marahiel and co-workers (Stachelhaus et al., 1999) defined a signature sequence consisting of ten amino acids lining with the ten residues of the phenylalanine-specific binding pocket located at positions 235, 236, 239, 278, 299, 301, 322, 330, 331 and 517 in the GsrA (Phe) sequence (accession number: P14687). The model proposed by Townsend and co-workers (Challis et al., 2000) uses only the first eight of these critical residues.
Preliminary specificity assignments of albicidin synthase AlbI, AlbIV, AlbVII and AlbIX NRPS modules were made by comparison of complete sequences between conserved motifs A4 and A5 with sequences in the Genbank database. The corresponding sequence of the AlbIV NRPS-5 module is most related to domain 5 of bacitracin synthase 3 (BA3) from B. licheniformis that was suggested to activate Asn (Konz et al., 1997). Corresponding sequences of AlbI and AlbIX NRPS-1, NRPS-3, NRPS-6 and NRPS-7 modules, apart from their very high similarity with XabB, exhibited the highest degree of overall identity (39%) with the Blm NRPS2 module of the biosynthetic gene cluster for bleomycin from S. verticillus that specifies for β-Alanine (Du et al., 2000). The corresponding sequence of AlbVII PKS-4 produced the highest significant alignment with acetate-CoA ligase from Sulfolobus solfataricus (Genbank accession number: AAK41550), aryl-CoA ligase from Comamnonas testosteroni (Genbank accession number: AAC38458) and 4-hydroxybenzoate-CoA ligase from R. palustris. The sequence between motifs A4 and A5 of the AlbI NRPS-2 could not be significantly aligned with any sequence present in the Genbank database. Comparison of this sequence with the corresponding sequence of GrsA (Phe) revealed that parts of the putative core and structural “anchor” sequences of AlbI NRPS-2 are deleted (FIG. 5), suggesting that the AlbI NRPS-2 substrate binding pocket is not functional. In the Figure, amino acids of the six Alb NRPSs and of Alb PKS4 that are identical or similar to GrsA or Blm sequences (A=G; D=E; I=L=V; R=K) are shown in bold. Amino acids underlined in the GsrA sequence correspond to the phenylalanine-specific binding pocket. The positions of these amino acids in the GrsA primary sequence are indicated at the top of the figure. Amino acids underlined in the other sequences correspond to putative constituents of binding pockets, aligned with the seven residues of the phenylalanine-specific binding pocket of GrsA. Shaded amino-acids correspond to the putative core sequences and structural “anchors” based on comparison with the GrsA binding-pocket structure.
Alignment of the primary sequence between conserved motifs A4 and A5 of the AlbI, AlbIV, AlbVII and AlbIX NRPS-1, NRPS-3, NRPS-5, NRPS-6, NRPS-7 and PKS4 modules with the corresponding sequence of GrsA (Phe) (FIG. 5) revealed the putative constituents of binding pockets that constitute the codes as defined by Marahiel and co-workers (Stachelhaus et al., 1999). These codes were compared with those of proteins most related to the sequence between the A4 and A5 motifs (Table 8) and were analyzed with the model proposed by Townsend and co-workers (Challis et al., 2000, jhunix.hcfjhu.edu/ ̃ravel/nrps//). Using these codes, we were able to predict the Asparagine specificity of the AlbIV NRPS-5 module. The AlbIV NRPS-5 signature is 100% identical to BacC-M5 (Asn) and TyrC-M1 (Asn) codes identified in bacitracin synthetase 3 from B. licheniformis and in tyrocidine synthetase 3 from B. brevis (Table 8). The AlbIV NRPS-5 signature is also identical to the Asn code defined by Marahiel and co-workers (1997), except that I is replaced by L at position 299 (Table 8). The AlbI and AlbIX NRPS-1, 3, 6 and 7 signatures did not match any of those defined by Marahiel and co-workers (1997). Similarly, convincing predictions using the model proposed by Townsend and co-workers were not obtained either (Challis et al., 2000, jhunix.hcfjhu.edu/ ̃ravel/nrps//). The AlbI and AlbIX NRPS-1, 3, 6 and 7 signatures diverged from all NRPS signatures previously described, except from the XabB signature that is identical to the AlbI NRPS-1 and 3 signatures. The signature most closely related to AlbI NRPS-1 and 3 specify Pro and the signature most closely related to AlbIX NRPS-6 and 7 specify Ser, but the degree of similarity in both cases is very weak (Table 8). The PKS-4 signature is similar to the AlbI NRPS-1 and NRPS-3 signatures at positions 235, 299 and 301.
Analysis of alignment of the primary sequence between conserved motifs A4 and A5 of the AlbI and AlbIX NRPS-1, NRPS-3, NRPS-6 and NRPS-7 modules with the corresponding sequences of the bleomycin synthase (Blm) NRPS2 (β-Ala) and gramicidin S synthetase (GrsA) modules (FIG. 5) revealed that (i) sequences of AlbI NRPS-1 and AlbI NRPS-3 differ only at the level of two residues that are not involved in substrate binding, (ii) sequences of AlbIX NRPS-6 and AlbIX NRPS-7 are 100% identical, (iii) sequences of AlbI NRPS-1 and AlbI NRPS-3 are very similar to sequences of AlbIX NRPS-6 and AlbIX NRPS-7 but differ at the level of five putative constituents of binding pocket, (iv) AlbI and AlbIX NRPS residues, which are similar to residues of Blm NRPS2 (β-Ala) or GrsA (Phe), are essentially located at the level of the putative core sequences and structural “anchor”, and differ at the level of putative constituents of the binding pocket.
Binding-pocket constituents forming the NRPS signatures have been classified into three subgroups according to their variability among 160 specificity-conferring signature sequences (Stachelhaus et al., 1999): (i) invariant residues Asp235 and Lys517 that mediate key interactions with the α-amino and α-carboxylate group of the substrate, respectively; (ii) moderately variant residues in positions 236, 301 and 330 which correspond to aliphatic amino acids and which may modulate the catalytic activity and fine-tune the specificity of the corresponding domains; (iii) highly variant residues in positions 239, 278, 299, 322 and 331 which may facilitate substrate specificity. AlbI and AlbIX NRPS-1, 3, 6 and 7 signatures are not totally in accordance with this classification. Invariant residue Lys517 is conserved in the four NRPS signatures, indicating the presence of an α-carboxylate group in the corresponding substrates. The Asp235Ala alteration is not consistent with an α-amino acid substrate. Birch and co-workers (Huang et al., 2001) assumed that the initial alanine residue in the XabB signature was consistent with a nonproteinogenic hydroxy acid substrate by analogy with the initial glycine in the signature of the hydroxyisovaleric-acid (HVCL) loading domain of enniatin synthetase. The presence of an initial Alanine in the AlbVII PKS-4 signature (FIG. 8) and in several 4-hydroxybenzoate-CoA ligase codes may confirm this hypothesis. However, the HVCL loading domain of enniatin synthetase (Table 8) and AlbVII PKS4 are not preceded by a C domain and are not followed by a PCP domain, in contrast to the AlbI and AlbIX NRPS-1, 3, 6 and 7 modules. An Asp235Val alteration was recently described in the β-Ala specificity-conferring code (Du et al., 2000, Table 8), suggesting that the substrate of AlbI and AlbIX NRPS-1, 3, 6 and 7 modules may be different from α-amino acids but may contain an amino group. Residue 236 is an aliphatic residue (Val or Ile) in all AlbI and AlbIX NRPS-1, 3, 6 and 7 signatures. Residue 301 is an aliphatic residue (Ala) in the AlbI NRPS-1 and 3 codes, but it is a Ser in the AlbIX NRPS-6 and 7 signatures. Residue 330 is not an aliphatic residue in the four NRPS signatures but an Asp. Similar alterations are present in the β-Ala code: residue 236 is an Asp, residue 301 is a Ser and residue 330 is an aliphatic amino acid. Concerning highly variable residues, AlbI NRPS-1 and 3 signatures differ from AlbIX NRPS-6 and 7 signatures at residue positions 299, 322 and 331, confirming that both types of NRPS modules specify different substrates.
Table 8: Comparison of signature sequences, as defined by Marahiel and co-workers (Stachelhaus et al., 1999), derived from sequences between the A4 and A5 domains of the AlbI, AlbIV, and AlbIX NRPS modules with those of Tyr-M1 (Pro) (Tyrocidine synthetase 2 module 1, accession number: AAC45929), VirS (Pro) (Virginiamycin S synthetase, accession number: CAA72310), HVCL (hydroxyisovaleric acid-CoA ligase, ACL1 enniatin synthetase, accession number: S39842), EntF-M1 (Ser) (Enterobactin synthase, accession number: AAA92015), β-Ala code (β-Ala selectivity-conferring code defined by Du et al., 2000), BacC-M5 (Asn) (Bacitracin synthetase 3, accession number: AAC06348), TyrC-M1 (Asn) (Tyrocidine synthetase 3, accession number: AAC45930) and Asn code (Asn selectivity-conferring code defined by Marahiel and co-workers (Stachelhaus et al., 1999). Amino acids of AlbI NRPS-1 and NRPS-3 signatures identical or similar to TyrB-M1 (Pro), VirS (Pro) and HVCL signatures (A=G; D=E; I=L=V; R=K) are shown in bold. Amino acids of AlbIX NRPS-6 and NRPS-7 signatures identical or similar to Vir (Pro) and Blm (&Ala) signatures (A=G; D=E; I=L=V; R=K) are shown in bold. Variability: 0 indicates invariant residues, +/−moderately variant residues and ++ highly variant residues.
EXAMPLE 18
Identification of Putative Promoters and Putative Terminators in XALB1
Putative rho independent terminators were identified downstream from albIV and albXVI using the Terminator program (Brendel and Trifonov, 1984), run with the Wisconsin PackageTM GCG software (FIG. 6). In the Figure, dashes indicate palindromic sequences. Symbols used in the Figure are: P, Primary structure value of putative terminator (minimum threshold value of 3.5 represents 95 percent of known, factor-independent, prokaryotic terminators); S, Secondary structure value of putative terminator. The presence of these terminators confined the proposed genetic organization of operons 1 and 3. A rho-independent terminator was identified in the intergenic region between albXVII and albXVIII, suggesting that the group of genes initially supposed to be organized in operon 4 may be in fact organized in two operons, operon 4 formed by albXVII and operon 5 by albXVIII B albXX. No putative rho independent terminator was found downstream from albIX and from albXX.
The 236 bp region between albI (operon 1) and albV (operon 2) is 100% identical to the sequence between xabB and thp genes that is assumed to contain a bidirectional promoter (Huang et al., 2000a and 2001), suggesting that transcription of operon 1 and 2 is regulated by the same bidirectional promoter region (Huang et al., 2001).
The 412 bp region comprised between albX (operon 3) and albXVII (operon 4) also contains a putative bidirectional promoter (FIG. 7). In the Figure, the sequence of putative promoters are underlined, and putative ATG or TTG start codons are in bold. The closest matches (TTGACA-18x-TATAGT) to the consensus-35 (TTGACA) and -10 (TATAAT) sequences for E. coli σ70 promoters occurs 61 bp upstream from albX (operon 3). The closest matches (TTCAGA-19x-TATACA) to the consensus sequences for E. coli σ70 promoters occur 320 bp upstream from albXVII (operon 4). The region between albXVII and albXVIII lacks any apparent E. coli σ70 promoter. However, the sequence immediately upstream from albXIX, corresponding to the coding sequence of albXVIII, potentially contains an unidirectional promoter (FIG. 7). The closest match (TTGCTC-19x-TATATT) to the consensus sequences for E. coli σ70 promoters occurs 33 bp upstream from albX. The presence of a terminator downstream from albXVII and of a promoter upstream from albXIX suggests that albXVIII is not transcribed and that albXIX and albXX form operon 5.
EXAMPLE 19
Cloning of the XALB2 Gene Cluster
The 6 kb EcoR I fragment carrying Tn5 and flanking sequence from strain AM37 was cloned in pBR325 and the obtained plasmid was designated pAM37 (Table 1). A 1.1 kb Hind III-Hind III DNA fragment from pAM37, named PR37 (Table 1), was labeled with 32P and used to probe the 845 clones from the genomic library of X. albilineans strain Xa23R1, previously described (Rott et al., 1996). Eight new cosmids hybridized to this probe and restored albicidin production in mutant AM37. One of these cosmid, pALB389, carrying an insert of about 37 kb (Table 1), was used for complementation studies of the five mutants not complemented by pALB540 and pALB571. Cosmid pALB389 complemented mutants AM10 and AM37. Mutant AM10 was initially thought to be complemented by pALB639 (Rott et al., 1996). However, further complementation studies showed that mutant AM10 was not complemented by pALB639 and that only three mutants (AM12, AM13 and AM36) were complemented by pALB639 containing the third genomic region XALB3 involved in albicidin production. A 3 kb EcoRI-EcoRI DNA fragment from pALB389 that hybridized with probe PR37 was sub-cloned in pUFR043 (Table 1). The resulting plasmid pAC389.1 complemented mutants AM10 and AM37, confirming that the second region involved in albicidin production, XALB2, was present in the 3 kb insert of pAC389.1.
EXAMPLE 20
Cloning of the XALB3 Gene Cluster
Cosmid pALB639, carrying an insert of 36 kb (Rott et al., 1996; Table 1) was used as a probe to compare the EcoRI restriction profiles of X. albilineans strain Xa23R1 with those of mutants AM12, AM13 and AM36 which were supposed to be mutated in the XALB3 gene cluster. An 11 kb band which was found in strain Xa23R1 but not in the three mutants was supposed to contain the XALB3 gene cluster. A 9.7 kb EcoRI DNA fragment purified from cosmid pALB639 also used as a probe in Southern blot analyse revealed the same 11 kb band. This 9.7 kb EcoRI DNA fragment was sub-cloned in pUFR043 (Table 1) and the resulting plasmid pAlb639A complemented mutants AM12, AM13 and AM36. The third region involved in albicidin production, XALB3, was therefore present in the 9.7 kb insert of pAlb639A.
EXAMPLE 21
Sequencing of the Tn5 Insertional Site of tox′ Mutants Located in XALB2 and XALB3 and Sequencing of the Genomic Regions XALB2 and XALB3
In FIG. 8, E, H, Sa and S indicate restriction endonuclease cut sites for EcoRI, HindIII, SalI and Sau3AI, respectively. The DNA inserts carried by plasmids pAC389.1, pALB639A or pEV639 are represented by the bars at the top of the respective figures. Positions of the Tn5 insertional sites of mutants AM10, AM12, AM36 and AM37 were determined by sequencing and are indicated by vertical arrows. The DNA region corresponding to the Tn5 flanking regions in pAM10, pAM12.1, pAM36.2 and pAM37 and in the PR37 DNA fragment are represented by the bars at the bottom of the respective figures. The location and direction of albXXI and albXXII are indicated by thick black arrows. The location of other orfs in XALB2 similar to those described by Huang et al. (2000b) are indicated by thick white arrows.
The 7 kb EcoR I fragment carrying Tn5 and flanking sequence from strain AM10 was cloned in pBluescript II KS (+), and the obtained plasmid was designated pAM10 (Table 1). The sequences between EcoRI sites and the Tn5 insertional site of mutants AM10 and AM37 were sequenced from the resulting plasmids pAM10 and pAM37, respectively. The complete double-strand nucleotide sequence of the 2,986 bp EcoR I B EcoR I insert of pAC389.1 was determined from sequencing results of plasmids pAC389.1, pAM10 and pAM37 (FIG. 8). The Tn5 insertional sites of mutants AM10 and AM37 were sequenced from plasmids pAM10 and pAM37 (Table 1), respectively, using the sequencing primer GUSN (5′tgcccacaggccgtcgagt3′) that annealed 135 bp downstream from the insertional sequence IS50L of Tn5-gusA. The Tn5 insertional site of AM10 and AM37 was located at position 2107 and 1882, respectively.
The EcoRI fragments carrying Tn5 and the flanking sequences from mutants AM12 and AM36 were cloned in pBR325 (Rott et al., 1996; Table 1). The sequences between EcoRI site and the Tn5 insertional site of mutants AM12 and AM36 were sequenced from the resulting plasmids pAM12.1 and pAM36.2, respectively. The complete double-strand nucleotide sequence of the 9,673 bp EcoR I B Sau3A I insert of pALB639A was determined from the sequencing results of plasmids pAM12.1, pAM36.2 and pALB639A (FIG. 8). The Tn5 insertional site of mutants AM12 and AM36 was sequenced from plasmids pAM12.1, pAM36.2 using the sequencing primer GUSN (5′tgcccacaggccgtcgagt3′) that annealed 135 bp downstream from the insertional sequence IS50L of Tn5-gusA. The Tn5 insertional site of AM12 and AM36 was located at position 6500 and 7232, respectively (FIG. 8).
EXAMPLE 22
Homology Analysis and Genetic Organization of XALB2 (FIG. 8)
The sequence of 2986 bp containing XALB2 is 99.4% identical to the sequence of 2989 bp containing xabA described in X. albilineans strain LS155 from Australia (Huang et al., 2000b; accession number AF191324). The Tn5 insertional site of mutant LS156 described in xabA is 15 bp upstream from the insertional site of AM37. The orf disrupted in AM37 and AM10, designed albXXI, is identical to xabA except a C which replaces a T at position 1642. albXXI potentially encodes a protein of 278 aa with a predicted size of 29.3 kDa which is 100% identical to the potential product of xabA, described as a phosphopantetheinyl transferase (Huang et al., 2000b). Region XALB2 contains three additional orfs (orf1, orf2, and orf3) similar to those described by Huang et al., (2000b; orf, rsp6 and aspT). orf2 and orf3 are 100% identical to rsp6 and aspT respectively, and orf1 is similar to but smaller than orf. There are no close matches to the E. coli γ70 promoter B10 (TATAAT) and B35 (TTGACA) consensus sequence, and no putative RBS site upstream from the putative start codon ATG of albXXI. The putative factor-independent transcription site described at 42 bp downstream from the TGA stop codon of xabA (Huang et al., 2000b) is also present at the same position downstream from albXXI.
EXAMPLE 23
Homology Analysis and Genetic Organization of XALB3 (FIG. 8)
The orf disrupted in mutants AM12 and AM36 was located between nucleotide 6090 (ATG) and 8009 (TAA) and was designed albXXII. The first ATG at position 6090 is not preceded by a putative ribosome binding sequence, suggesting that the start codon is the ATG at position 6105 which is preceded at position B9 by the putative ribosome binding site sequence GGAG. A putative rho independent terminator was identified at position 8082, 73 b downstream from albXXII (FIG. 6). There are no close matches to E. coli σ70 promoter B10 (TATAAT) and B35 (TTGACA) consensus sequence upstream from the putative start codon. The SalI DNA fragment corresponding to DNA sequence from nucleotide 5510 to nucleotide 8124, which contains the 595 bp upstream from the putative start codon, the orf albXXII and the putative rho independent terminator, was sub-cloned in pUFR043 in the opposite direction to LacZ (Table 1). The resulting plasmid pEV639 (Table 1) complemented mutants AM12, AM13 and AM36, confirming that (i) the third region involved in albicidin production, XALB3, was present in the insert of pEV639; (ii) albXXII is not transcribed as part of a larger operon; and (iii) the 595 bp upstream the putative start codon contain a promoter.
The potential product of albXXII, designated AlbXXII, is a protein of 634 aa with a predicted size of 71.5 kDa. This protein is very similar to the heat shock protein HtpG from Pseudomonas aeruginosa (identities=82%) and from Escherichia coli (identities=60%) (Table 4). The methionine encoded by the putative start codon at position 6105 was aligned with the first aminoacid of the heat shock protein HtpG from Pseudomonas aeruginosa, confirming that albXXII initiates at position 6105.
Complementation of Tox− Mutants with the albXXII Gene in Fusion with LacZ
A 1,948 bp fragment corresponding to the entire 1,903 bp orf of albXXII and flanking nucleotides was PCR amplified from cosmid pALB639 with the forward primer 5′tttgaattcgcacctaccgatgagcgtgg3′ and the reverse primer 5′tttggatccgtgcgtcactgcttacgccg3′. Convenient in frame-EcoRI and BamHI restriction sites for further cloning were simultaneously introduced with forward and reverse PCR primers, respectively. The PCR fragment was cloned into pGEMT vector (Promega) and sequenced. Several clones of the resulting plasmid pGemT/albXXII were sequenced. Because several PCR derived point mutations were observed in all the sequenced clones, a 1,920 bp BglII-SalI fragment from pEV639 (corresponding to the 1,809 5′ terminal nucleotides of albXXII orf plus 111 bp downstream the stop codon) was cloned into a pGemT/albXXII clone between the BglII site located at position 94 of the albXXII orf and the SalI site of the vector's multiple cloning site. The resulting plasmid pGemT/albXXIIbis contained an intact albXXII orf that was then subcloned as an EcoRI-SalI fragment into pUFR043 to generate pEValbXXII. This construct of albXXII in fusion with LacZ was transferred by triparental conjugation into Xa23RI insertion mutants. pEValbXXII complemented mutants AM12, AM13 and AM36 (see table 9). These results confirmed that (i) the third region involved in albicidin production, XALB3, was present in the insert of pEValbXXII; and (ii) albXXII is not transcribed as a part of a larger operon.
Complementation of Tox− Mutants with the htpG Gene from E. coli 
A 2,343 bp fragment corresponding to the htpG gene of E. coli plus 458 bp downstream the stop codon was PCR amplified from purified DH5α genomic DNA with forward primer 5′tttgaattccatgaaaggacaagaaactcgtgg3′ and reverse primer 5′gcctgcggaatggtacgcgggaagccgtcc3′. A convenient in frame-EcoRI restriction site was introduced with the forward PCR primer. The PCR fragment was cloned using the pGEMT vector system (Promega). Three resulting clones potentially containing plasmid pGemT/HtpG were sequenced, and one clone containing the correct sequence was selected. The 2,343 bp PCR insert was then subcloned as an EcoRI-SalI fragment into pUFR043 to generate pEVHtpG, the SalI site corresponding to the site of the vector's multiple cloning site. This HtpG gene, in fusion with the LacZ construct, was able to restore albicidin production after transfer by triparental conjugation into AM12, AM13 and AM36 Xa23RI mutants. This result is i/ further evidence of the involvement of a molecular chaperone HtpG in the biosynthesis of albicidin (table 9), ii/ the first report of the requirement of a molecular chaperone HtpG in NRPS and PKS metabolism.
EXAMPLE 24
Heterologous Production of Albicidin in Fast Growing Xanthomonas axonopodis pv. Vesicatoria 
This example illustrates the construction of a heterologous expression system harboring the three XALB regions, its transfer into a fast growing host, Xanthomonas axonopodis pv. vesicatoria and the subsequent production of a potent toxin with an antibiotic activity similar to that of albicidin. This work is a milestone in the validation of the albicidin biosynthesis model because it gives experimental evidence that the entire biosynthetic machinery required for albicidin biosynthesis has been identified, cloned, sequenced and transferred into an heterologous host, driving the production of albicidin. Cosmid pALB571 which covers the complete sequences of operons 1 and 2 was used to transfer operons 1 and 2 (FIG. 1). Operons 3 and 4 (from pALB540), XALB2 (from pAC389.1) and XALB3 (from pEV639) were subcloned into a single plasmid, pOp3-4/XALB2-3 (see below). Plasmid pOp3-4/XALB2-3 derived from shuttle vector pLAFR3 that carries one selective gene for resistance to tetracyclin and that belongs to incompatibility group IncP (Table 1). Cosmid pALB571 derived from shuttle vector pUFR043 that carries two selective genes for resistance to kanamycine and gentamycine and that belongs to incompatibility group IncW (Table 1).
Sub-cloning of Operons 3 and 4 and XALB2 and XALB3 Regions into a Single Plasmid (FIG. 12).
A 2,787 bp BamHI-PstI fragment from pALB540, corresponding to a portion of operon 4, was subcloned into pBCKS(+), yielding pBC/Op4Δ (step 1). A XhoI site was introduced into this vector immediately upstream from the BfrI site by directed mutagenesis. Mutagenesis was performed with primers XhoIAlb anticodant 5′cgccttaagcagctcgagtagactgcaatc3′ and XhoIAlbcodant 5′gattgcagtctactcgagctgcttaaggcg3′ and yielded plasmid pBC/Op4ΔXhoI (step 2). The 2,986 bp EcoRI fragment from pAC389.1 (containing XALB2) was then subcloned into pBC/Op4ΔXhoI, yielding pBC/Op4Δ/XALB2 (step 3). A 10,762 bp BfrI fragment from pALB540 and containing complete operon 3 and the beginning of operon 4 was subcloned into pBC/Op4Δ/XALB2 yielding pBC/Op3-4/XALB2 (step 4). The 2,615 bp SalI fragment from pEV639 (containing XALB3) was subcloned into pBKS, yielding pBKS/XALB3 (step 5). The SalI site located on the KpnI side of the polylinker was then destroyed and substituted by a XhoI restriction site by directed mutagenesis. This mutagenesis was performed with primers XhoSalXaHTPGR 5′gcttatcgataccctcgaggaaggcgatatcg3′ and XhoSalXaHTlPGF 5′cgatatcgccttcctcgagggtategataagc3′, yielding pBKS/XALB3XhoI (step 6). Finally, the XhoI cassette of pBC/Op3-4/XALB2 was subcloned into the SalI restriction site of pBKS/XALB3XhoI, yielding pBKS/Op3-4/XALB2-3 (step 7). This construct harbours an XhoI cassette containing complete operons 3 and 4 from XALB1, albXXI from XALB2 and albXXII from XALB3. An XhoI site was added to the BamHI site of the pLAFR3 shuttle vector polylinker using the adaptor AdApTBamHIXhoI 5′gatcgctcgagc3′, yielding pLAFR3XhoI (step 8). The XhoI cassette from pBKS/Op34/XALB2-3 was then cloned into pLAFR3XhoI yielding pOp3-4/XALB2-3 (step 9). This last construct was used, along with pALB571 (operons 1 and 2), for heterologous expression of albicidin in X. axonopodis pv. vesicatoria. 
Albicidin Production Assays
The four combinations of plasmids (i.e. pUFR043-pLAFR3, pUFR043-pOp34/XALB2-3, pAlb571-pLAFR3 and pAlb571-pOp3A4XALB2-3) were transferred into X. axonopodis pv. vesicatoria strain Xcv 91-11BR1 by triparental mating. Exconjugant clones resistant to tetracycline and kanamycin were isolated. Assays for albicidin production were performed with these exconjugants clones using the same method described in Example 1 except that tetracycline (12 mg/ml) and/or kanamycin (50 mg/ml) were added to SPA medium. Tetracycline and kanamycin resistant E. coli clones, DH5αKT and DH5αAlbrKT (Table 1), were used as tester strains to evaluate albicidin production to ensure that growth inhibition was not due to the presence of these two antibiotics in SPA medium. Both clones, DH5αKT and DH5αAlbrKT, are tetracycline and kanamycin resistant because they carry plasmids pLAFR3 and pUFR043. The albicidin resistant DH5αAlbrKT clone derived from strain DH5αAlbr (Table 1) which is a spontaneous albicidin resistant clone isolated in a growth inhibition zone produced by X. albilineans strain Xa23R1.
Without antibiotics in the SPA medium, growth of clones DH5αKT and DH5αAlbrKT was not inhibited in all assays performed with the different X. axonopodis pv. vesicatoria exconjugants. Surprisingly, when kanamycin was present in the SPA medium, growth of both DH5αKT and DH5αAlbrKT was inhibited in all assays performed with the X. axonopodis pv. vesicatoria exconjugants. These results suggested that, in the presence of kanamycin, all X. axonopodis pv. vesicatoria exconjugants produced an antibiotic inhibiting growth of E. coli. Because exconjugants containing only empty vectors (pUF043 and pLAFR3) induced inhibition of E. coli, this antibiotic did not result from the expression of XALB1, XALB2 and/or XALB3. Additionally, there was no cross resistance between this antibiotic and albicidin. When tetracyclin was present in the bioassay medium, but not kanamycin, growth of the albicidin resistant clone (DH5αAlbrKT) was not inhibited by any of the exconjugants. In contrast, growth of the albicidin susceptible E. coli strain (DH5αKT) was inhibited by the exconjugants harbouring pALB571 and pOp34/XALB2-3 plasmids, but not by exconjugants harbouring the other three combinations of plasmids (Table 10). This result suggested that expression of the XALB1, XALB2 and XALB3 regions in X. axonopodis pv. vesicatoria (harbouring pALB571 and pOp34/XALB2-3 plasmids) led to the production of an albicidin-like antibiotic. This product inhibited growth of an albicidin sensitive E. coli (DH5αKT) and had no effect on the growth of an albicidin resistant clone (DH5αAlbrKT).
Preliminary results indicated that pLAFR3 derived plasmids were relatively unstable in the absence of tetracycline in the culture medium, suggesting that genes carried by pOp3-4×ALB2-3 were not expressed when X. axonopodis pv. vesicatoria exconjugants pALB571/pOp34/XALB2-3 were grown without tetracycline. Consequently, these exconjugants did not produce the albicidin-like compound in absence of any antibiotic in the culture medium (Table 10). Preliminary results also indicated that pUFR043 derived plasmids are relatively stable in X. axonopodis pv. vesicatoria in absence of antibiotic selection, suggesting that genes carried by pALB571 are expressed when X. axonopodis pv. vesicatoria exconjugants pALB571/pOp34/XALB2-3 were grown on media without kanamycin. Consequently, these exconjugants produced the albicidin-like compound on SPA containing only tetracyclin.
Two E. coli DH5αKT clones, that spontaneously grew within the growth inhibition zone of a X. axonopodis pv. vesicatoria pALB571-pOp3-4/XALB2-3 exconjugant on SPA+tetracycline medium, were isolated and tested for resistance to albicidin. No growth inhibition was observed when these clones were used as tester strains in an albicidin production assay performed with X. albilineans Xa23R1. These results showed that cross-resistance occurs between the albicidin-like product of X. axonopodis pv. vesicatoria and albicidin produced by X. albilineans, suggesting that both molecules are similar. Comparison of chemical characteristics of the two molecules will, however, be necessary to confirm that the two molecules are identical.
The invention includes the isolation and sequencing of a region of 55,839 bp from X. albilineans strain Xa23R1 containing the major gene cluster XALB1 involved in albicidin production. Analysis of this region allowed us to predict the genetic organization of the gene cluster XALB1 which contains 20 ORFs grouped in four or five operons (FIG. 1). Because albXVII is a truncated gene, XALB1 genes may be organized in five operons. Therefore, we will from now on consider albXVII as part of operon 4 and albXIX and albXX as part of operon 5. Similar operon-type organizations for antibiotic biosynthesis clusters are well known and have been postulated to facilitate cotranslation of genes within the operon to yield equimolar amounts of proteins for optimal interactions to form the biosynthesis complexes (Cane, 1997). Overlapping genes involved in the same process are also quite common in bacteria (Normark et al., 1983).
Previous results of transposon mutagenesis and complementation studies (Rott et al., 1996; Rott, unpublished results) are in accordance with the predicted genetic organization of XALB 1 described in this study, and allowed us to establish that operons 1, 2 and 3 are involved in albicidin biosynthesis: (I) Tox− mutants with a Tn5-gusA insertion site located in DNA fragments B, C, G and D were complemented by cosmid pALB571 and not by cosmid pALB540, confining that cosmid pALB571 potentially contains the entire operon 1; (ii) Tox− mutants with a Tn5-gusA insertion site located in DNA fragments A and H were complemented by both cosmids pALB540 and pALB571, confirming that both cosmids potentially contain the entire operon 2; (iii) mutant XaAM1 with a Tn5-gusA insertion site located in DNA fragment J is the only Tn5 Tox− mutant complemented by cosmid pALB540 and not by cosmid pALB571, confirming that cosmid pALB540 potentially contains the entire operon 3. Our mutagenesis studies did not confirm that operons 4 and 5 are required for biosynthesis of albicidin. The para-aminobenzoate (PABA) is required for the growth of many bacteria probably including X. albilineans, suggesting that a mutation in albXVII may be lethal and explaining why we did not obtain any mutant disrupted in this gene.
Putative bidirectional promoters were identified between operons 1 and 2 (Huang et al., 2001) and between 3 and 4 (FIG. 7), confirming the prediction of genetic organization of XALB1. The region upstream from operon 1 is 100% identical to the region upstream from the xabB start codon which was described as a functional promoter during the phase of albicidin accumulation in Australian strain Xa13 of X. albilineans (Huang et al., 2001). Involvement of several operons in albicidin biosynthesis suppose that they are transcribed during the same time. Promoter activities of regions upstream from putative operons 2, 3, 4 and 5 need to be determined to precise if these promoters are functional during the same growth phase of X. albilineans as the promoter upstream from operon 1.
Potential rho-independent transcription terminators were identified downstream from operons 1, 3 and 4 (FIG. 6) confirming prediction of the genetic organization of these three operons. Because operons 2 and 5 are convergent (FIG. 1) and separated by a very short region of 22 bp between albIX and albXX, stop codons may allow transcription termination in the absence of sequences corresponding to potential rho-independent transcription terminators downstream from these operons. It is quite possible that simultaneous transcription of operons 2 and 5 involving the presence of two RNA polymerases (one on each strand of DNA) may cause RNA polymerases to pause at the end of each operon because of steric interference between both polymerase complexes in the same short region.
The presence of putative RBSs upstream of the ATG start codons of all ORFs, except for albXVIII, suggests that these ORFs are translated in X. albilineans. The absence of a canonical RBS upstream from albXVIII further indicates that this ORF is probably not expressed. GTG and TTG codons (usually valine and leucine codons) generally serve as procaryotic start codons when located near the 5′ end of an RNA message, but GTG start codons were also described far from the 5′ end of messenger RNA in the bacitracin biosynthesis cluster of B. lucheniformis (Genbank Accession No. AF184956) or in the bleomycin biosynthetic gene cluster of S. verticillus (Genbank Accession No. AF210249). This is in accordance with the fact that the two potential TTG start codons are the first start codons in operons 1 and 4 of XALB1, and that the two potential GTG start codons initiate internal cistrons. The albI and albXVII genes, like xabB (Huang et al., 2001), use TTG as a start codon, which may impose post-transcriptional control of the rate of gene product formation (McCarthy and Gualerzi, 1990).
The predicted genetic organization of operons 1 and 2 presents similarities with the organization of the region involved in albicidin production in strain Xa13 of X. albilineans from Australia (Huang et al. 2000a, Huang et al., 2001). This latter region also contains two divergent operons involved in albicidin production, one comprising the xabB gene (similar to albI, but with a large deletion) and the xabC gene (100% identical to albII) and the other containing thp gene (100% identical to albV). In addition, the sequence between the two operons in stain Xa13 is 100% identical to the sequence between operons 1 and 2, indicating that both clusters are controlled by the same bidirectional promoter. However, transposon mutagenesis studies of Xa13 showed no evidence of another cistron downstream of xabC that may be involved in albicidin production (Huang et al., 2000a), suggesting that the Xa13 xab operon differs from the Xa23R1 operon 1, which contains two additional genes downstream from albII that are potentially involved in albicidin production (albIII and albIV; refer FIG. 1).
Homology analysis revealed that four NRPS and/or PKS genes are present in XALB1 (FIG. 2), and these genes may be involved in the biosynthesis of the albicidin polyketide-polypeptide backbone (albI, albIV, albVII and albIV). NRPS and PKS enzymes are generally organized into repeated functional units known as modules, each of which is responsible for a discrete stage of polyketide or polypeptide chain elongation (Cane and Walsh, 1999). Each PKS or NRPS module is made up of a set of three core domains, two of which are catalytic and one of which acts as a carrier, and together are responsible for the central chain-building reactions of polyketide or polypeptide biosynthesis. Both PKS and NRPS core domains utilize analogous acyl-chain elongation strategies in which the growing chain, tethered as an acyl-S-enzyme to the flexible 20 Å long phosphopantetheinyl arm of an acyl carrier protein (ACP) or peptidyl carrier protein (PCP) domain, acts as the electrophilic partner that undergoes attack by a nucleophilic chain-elongation unit, a malonyl- or aminoacyl-S-enzyme derivative, respectively, itself covalently bound to a downstream ACP/PCP domain. In the case of a PKS, the fundamental chain-elongation reaction, a C—C bond-forming step, is mediated by a ketosynthase (KS) domain that catalyzes the transfer of the polyketide acyl chain to an active-site cysteine of the KS domain, followed by condensation with the methylmalonyl- or malonyl-S-ACP by a decarboxylative acylation of the malonyl donor unit. An additional essential component of the core PKS chain-elongation apparatus is an associated acetyltransferase (AT) domain, which catalyzes the priming of the donor ACP sidearm with the appropriate monomer substrate, usually methylmalonyl- or malonyl-CoA. The comparable core domains of an NRPS biosynthetic module function in a chemically distinct but architecturally and mechanistically analogous fashion. In the latter case, the key chain-building reaction, a C—N bond-forming reaction, involves the generation of the characteristic peptide bond by nucleophilic attack of the amino group of an amino acyl-S-PCP donor on the acyl group of an upstream electrophilic acyl- or peptidyl acyl-S-PCP chain, catalyzed by a condensation (C) domain. In functional analogy to the PKS AT domain, the core of the NRPS module utilizes an adenylation (A) domain to activate the donor amino-acid monomer as an acyl-AMP intermediate, which is then loaded onto the downstream PCP side chain. Both the AT and A domains of the respective PKS and NRPS modules act as important gatekeepers for polyketide or polypeptide biosynthesis, exhibiting strict or at least high specificity for their cognate malonyl-CoA, methylmalonyl-CoA or amino acid substrates. In addition to the basic subset of core domains, each PKS or NRPS also has a special set of dedicated domains responsible both for the initiation of acyl-chain assembly by loading of a starter unit onto the first, furthest upstream PKS/NRPS module, as well as a chain-terminating thioesterase (TE) domain, most often found fused to the last module, that is responsible for detachment of the most downstream covalent acyl enzyme intermediate and off-loading of the mature polyketide or polypeptide chain (Cane and Walsh, 1999).
XALB1 potentially encodes four PKS modules and seven NRPS modules. Most of the bacterial NRPS gene clusters described up to now are organized in operon-type structures, encoding multi modular NRPS proteins with individual modules organized along the chromosome in a linear order that parallels the order of amino acids in the resultant peptide, following the “colinearity rule” for the NRPS-template assembly of peptides from amino acids (Cane, 1997; Cane et al., 1998; Cane and Walsh, 1999; von Döhren et al., 1999). PKS and NRPS modules are apparently not organized according to this “colinearity rule” for albicidin biosynthesis because of the following features: (I) NRPS and PKS genes are expressed in two divergent operons; (ii) no AT domain was identified in PKS-2 and PKS-3 domains, suggesting involvement of a separate enzyme; (iii) the A domain of NRPS-2 is not functional, suggesting the involvement of a trans-acting A domain; (iv) a single chain-terminating TE domain was identified in XALB1 which may be responsible of the release of the full length albicidin polyketide-polypeptide backbone from the enzyme complexes. Exception to the “colinearity rule” has also been shown for the syringomycin synthetase of P. syringae (Guenzi et al., 1998), for the exochelin synthetase of Mycobacterium smegmatis (Yu et al., 1998) and for the bleomycin synthetases of S. verticillus (Du et al., 2000).
On the basis of the deduced functions of individual NRPS and PKS domains we have aligned the four PKS and the seven NRPS modules to suggest two different putative linear models for the synthesis of the albicidin polyketide-peptide backbone (FIG. 9). In the Figure, NRPS and PKS domains are abbreviated as follows: A, adenylation; ACP, acyl carrier protein; AL, acyl-CoA ligase; AT, acyltransferase; C, condensation; HBCL, hydroxybenzoate-CoA ligase; KR, ketoreductase; KS, ketoacyl synthase; PCP, peptidyl carrier protein. Asn designates asparagine. X1 and X2 indicate substrates incorporated by NRPS-1 and 3 and by NRPS-6 and 7, respectively. The crossed A domain in NRPS-2 indicates that this deleted domain may be not functional. In model 1, (FIG. 9A), (i) the PKS-1 module alone is responsible for the initiation of the acyl-chain assembly, (ii) PKS4 (HBCL) interacts with PKS-2 and PKS-3 as an AT domain to allow acyl transfer and (iii) NRPS-5 interacts with only NRPS-2. In model 2 (FIG. 9B) two different modules, PKS-1 and PKS-4, are responsible for this initiation step. Model 2 leads to the biosynthesis of four different polyketide-polypetide backbones; in this model (i) PKS-1 (AL) and PKS-4 (HBCL) are in competition for initiation of albicidin precursors; (ii) a separate AT enzyme (potentially AlbXIII) interacts with PKS-2 and PKS-3 to allow acyl transfer; (iii) NRPS-5 interacts with NRPS-2; and (iv) NRPS-5 and NRPS-6 are in competition for interaction with NRPS4.
Both models are based on the fact that PKS-1 contains the AL and ACP1 domains, and PKS-4 shows homology with the hydroxybenzoate-CoA ligases. In other PKS systems, an N-terminal AL domain is involved in the activation and incorporation of an 3,4-dihydroxycyclo hexane carboxylic acid, a 3-amino-5-hydroxybenzoic acid or a long-chain fatty acid as a starter (Aparicio et al., 1996; Motamedi and Shafiee, 1998; Tang et al., 1998; Duitman et al., 1999). PKS-4 may be also involved in the activation and incorporation of hydroxy-benzoate but this latter domain lacks any ACP or PCP domain, suggesting that PKS-4 is responsible for initiation of the acyl-chain assembly (FIG. 9B) onto one of the three ACP domains of AlbI (ACP1, ACP2 or ACP3). The 277 amino-acids preceding the PKS4 module in AlbVII may be necessary for the intercommunication between AlbVII and AlbI. The presence of two different PKS modules potentially involved in the initiation of the acyl-chain assembly suggests a competition of these two modules for the initiation of two different albicidin polyketide-polypeptide backbones, and this could contribute to the production of multiple, structurally related albicidins by the same cluster XALB1. Production of two different components, one initiated by PKS-4 containing an additional aromatic ring due to incorporation of hydroxybenzoate, may explain why partial characterization of albicidin indicated the presence of a variable number (three or four) of aromatic rings (Huang et al., 2001).
In AlbI, PKS-1 is followed by the PKS-2 module which contains a KS domain and a KR domain upstream from two ACP domains (ACP2 and ACP3) and it lacks any discernable AT domain. Tandem ACP domains are unusual within PKS modules but have been shown to occur in the biosynthesis of several fungal and bacterial polyketide synthases (Mayorga and Timberlake, 1992; Yu and Leonard, 1995; Takano et al., 1995; Albertini et al., 1995). However, the significance of the tandem ACP domains in these systems has not been solved yet. In our model 2, one of the tandem ACP (ACP2 or ACP3) may interact with PKS-4 for the initiation of an acyl-chain assembly (FIG. 9B). The absence of an AT domain in the PKS-2 module suggests that a separate AT domain is indispensable for the elongation of the acyl-chain initiated by this module. Separate AT enzymes encoded elsewhere in the genome were described in other systems for two PKS modules lacking AT domains: malonyl-CoA transacyclase gene (fenF) located immediately upstream from the B. subtilis PKS-NRPS mycA gene (Duitman et al., 1999) and an AT gene located 20 kb upstream from the M xanthus NRPS-PKS tal gene (Paitan et al., 1999). We have not identified an AT gene in the gene cluster XALB1 and in the two other genomic regions involved in albicidin production, XALB2 and XALB3, suggesting that the trans-acting AT gene may be encoded elsewhere in the genome. However, AlbXIII, which contains the motif GHSxG conserved in AT domains, may be potentially involved in the acyl transfer, but the similarity of AlbXIII with AT domains is not high enough to confirm this potential function of AlbXIII (FIG. 10). FIG. 10A describes alignment of the conserved motifs in AT domains from RifA-1, -2, -3, RifB-1, RifE-1 (Rifamycin PKSs, August et al., 1998) and BlmVIII (Bleomycin PKS; Du et al., 2000), identical amino acids are shown in bold. FIG. 10B describes alignment of AlbXIII (SEQ ID NO. 38), FenF (a malonyl-CoA transacylase located upstream from mycA, Duitman et al., 1999) and LipA (a lipase; Valdez et al., 1999); amino acids identical to conserved AT domains motifs are shown in bold.
AlbXIII contains only four of the eleven amino acids conserved in AT domains of rifamycin PKSs (August et al., 1998) and Bleomycin PKS (Du et al., 2000), and the AlbXIII sequence appears to be more closely related to lipases such as LipA (Valdez et al., 1999) rather than to AT domains (FIG. 10). However, FenF, the trans-acting AT domain involved in mycosubtilin biosynthesis, contains only seven of the eleven amino acids conserved in AT domains (Duitman et al., 1999; FIG. 10). AlbVII, that contains a HBCL domain, may be another candidate for the acyl transfer in PKS-2 (FIG. 9A) because HBCL exhibits some similarity with A domains at the level of cores A1, A2, A3, A4, A5 and A6 (Table 6). However, no HBCL involved in such a function has been described in the PKSs characterized so far.
In AlbI, PKS-2 is followed by the PKS-3 module which contains the KS2 and the PCP1 domains and it lacks any discernable AT or A domain. PKS-3 is located upstream from the NRPS modules and should therefore be involved in the linkage of polyketide and polypeptide moieties. The presence of a PCP domain in the PKS-3 module suggests the involvement of a trans-acting A domain rather than an AT domain. A putative candidate for this trans-acting A domain is the AlbIV NRPS-5 A domain because of the lack of a C domain in the AlbIV NRPS-5 module. However, by analogy with the BlmVIII PKS module, which is involved in the linkage of polypeptide and polyketide moieties of bleomycin and which contains an AT domain followed by a PCP domain (Du et al, 2000), the presence of a PCP is not incompatible with a possible interaction of the AlbI PKS-3 module with a separate AT domain. This latter trans-acting AT domain may be the same that interacts with the AlbI PKS-2 module, the AlbVII PKS-4 module, AlbXIII or an unindentified separate AT domain.
In AlbI, the PKS-3 module is followed by four NRPS modules. The NRPS-1, 2 and 3 modules display the ordered C, A and PCP domains, suggesting that they are involved in the incorporation of three amino acid residues. The A domain of the NRPS-2 module exhibits poor consensus at A2, A3, A5, A7, A8 A9 and A10 motifs and lacks completely the A6 motif (Table 6). In addition the NRPS-2 substrate binding pocket is partially deleted (FIG. 5). These features strongly suggest that the NRPS-2 A domain is inactive and that the loading of an amino-acid on the NRPS-2 PCP domain (PCP3) is possibly catalyzed by a trans-acting A domain as in HMWP1 (Gehring et al., 1998) and BlmIII (Du et al., 2000). A putative candidate for this trans-acting A domain is the NRPS-5 A domain present in AlbIV because of the lack of a C domain in NRPS-5 (FIG. 2). The additional sequence of 300 amino-acids present in the A domain of NRPS-5 may be necessary for the intercommunication between AlbI and AlbIV. As a consequence of the interaction between NRPS-2 and NRPS-5, a competition between PCP-3 and PCP-5 domains must occur to bind the amino acid activated by the NRPS-5 A domain. A similar competition between two PCP domains was described for syringomycin biosynthesis, during the interaction between SyrB, which contains A and PCP domains, and the last module of SyrE which contains C and PCP domains (Guenzi et al., 1998). The NRPS-4 module contains only a C domain which may transfer the intermediate products synthetized by AlbI to a PCP domain present in an other albicidin synthase. Similar transfers were described for mycosubtilin biosynthesis in which the MycA and MycB C-terminal C domains interact with the MycB and MycC N-terminal A domains, respectively (Duitman et al., 1999). Two different PCP domains may be involved in the transfer of the intermediate products synthetized by AlbI: the PCP-5 and PCP-6 domains which are present in the AlbIV NRPS-5 and AlbIX NRPS-6 modules, respectively. This possible competition between the two NRPS modules that contain two different A domains could also contribute to the production of multiple, structurally related albicidins by the gene cluster XALB1 (FIG. 9B). Because of the absence of a C-domain in the AlbIX NRPS-6 module, the intermediate product bound on the AlbIV PCP-5 domain would be necessarily transferred to the AlbIX PCP-7 domain, like the intermediate product bound on AlbIX PCP-6. AlbIX NRPS-7, which contains the single chain-terminating TE domain, may then be responsible for detachment of the mature albicidin polyketide-polypeptide backbone from the complex of enzymes.
The linear model 1 implies that operon 1 and operon 2 in X. Albilineans strain Xa23R1 from Florida potentially produce only one albicidin polyketide-polypetide backbone, with a competion at the level of ACP2/ACP3 and PCP3 and PCP5 which could explain the production by X. albilineans of compounds structurally related to albicidin (FIG. 9A). The linear model 2 implies that operon 1 and operon 2 in X. albilineans strain Xa23R1 from Florida potentially produce four different albicidin polyketide-polypetide backbones (FIG. 9B) because of (i) the competition of AL and HBCL domains for initation of acyl chain assembly and (ii) the competition of AlbIV NRPS-5 and AlbIX NRPS-6 modules for the incorporation of the next to last amino acid of the albicidin backbone. These four albicidin backbones may lead to the production of four components structurally very different The polyketide moieties of the acyl chains initiated by the AlbI AL domain or by the AbVII HBCL domain may be very different. The polyketide moiety of acyl chains initiated by the AlbVII HBCL domain may be shorter and may contain an additional aromatic ring. The presence of four structuraly different metabolites may explain the difficulty observed by Birch and Patil (1985a) to purify albicidin and to determine its chemical structure.
Homology analysis also revealed that AlbI NRPS-1 and 3 and AlbIX NRPS-6 and 7 specify unusual substrates which seem to contain an amino group and a carboxylate group but to be different from α-amino acids and β-alanine. Identification of several aromatic rings in albicidin (Huang et al., 2001) suggested that NRPS-1, -3, -6 and -7 are involved in incorporation of aromatic substrates. By analogy with the Asp235Val alteration in the β-Ala specificity-conferring code (Du et al. 2000), the Asp235Ala alteration in the NRPS-1, -3, -6 and -7 signatures could be consistent with a large distance between the amino group and the carboxylate group in the substrate specified by these modules. Based on this hypothesis, we suggest that operons 3, 4 and 5 are involved in the biosynthesis of two aromatic substrates: the para-aminobenzoate potentially synthesized by AlbXVII (para-aminobenzoate synthase), and the carbamoyl benzoate potentially synthesized by AlbXX (hydroxybenzoate synthase) and AlbXV (carbamoyl transferase). Incorporation of these nonproteinogenic substrates may explain why albicidin is insensitive to proteases (Birch and Patil, 1985a).
According to biosynthesis model 1 leading to the biosynthesis of only one polyketide-polypeptide albicidin backbone that may correspond to the major component produced by XAlb1, we propose a model allowing prediction of the composition and the structure of albicidin (FIG. 11). In the Figure, NRPS and PKS domains are abbreviated as follows: A, adenylation; ACP, acyl carrier protein; AL, acyl-CoA ligase; C, condensation; KR, β-ketoreductase; KS, β-ketoacyl synthase; PCP, peptidyl carrier protein. C atoms of albicidin-backbone are numbered 1 to 38. Bold methyl groups correspond to methylation of the albicidin backbone by AlbII or AlbVI. In this model, albicidin biosynthesis is initiated by loading of an acetyl-CoA by PKS-1 (step 1), and the chain product is elongated by incorporation of (I) malonyl-CoA by PKS-2 and PKS-3 (steps 2 and 3), (ii) para-aminobenzoate or carbamoyl benzoate by NRPS-1 and NRPS-3 (steps 4 and 6), (iii) asparagine by NRPS-2 coupled to NRPS-5 (step 5) and (iv) para-aminobenzoate or carbamoyl benzoate by NRPS-6 and NRPS-7 (steps 7 and 8). The presence of the KR domain in the PKS-2 module may lead to the formation of an hydroxyl group at the C2 atom of the albicidin backbone. This hydroxyl group might be methylated by AlbVI (O-methyltransferase). The acyl chain may also be modified by AlbII (C-methyltransferase) at C13 or C14.
The chemical composition (C40O15N6H35), the molecular weight (839), and the structure of the putative XALB1 product are in accordance with the partial characterization of albicidin published by Birch and Patil (1985a) which indicated that albicidin contains approximately 38 carbon atoms and a carboxylate group and that the molecular weight of albicidin was about 842. The presence of two ester linkages in our predicted albicidin structure is also in accordance with the fact that albicidin is detoxified by the AlbD esterase (Zhang and Birch, 1997). However, an unpublished albicidin analysis cited by Huang et al. (2001) indicated the presence of (I) two OCH3 groups and not one as in our predictive albicidin structure, (ii) one CN linkage and not eleven as in our predictive albicidin structure and (iii) a trisubstituted double bond that is not present in the putative XALB1 product.
In conclusion, homology analysis of XALB1 revealed unprecedented features for hybrid polyketide-peptide biosynthesis in bacteria involving a trans-action of four PKS and seven NRPS separate modules which could contribute to the production of multiple, structurally related polyketide-peptide compounds by the same gene cluster. Characterization of the full chemical structure of albicidin may be necessary to validate these models. Four NRPS modules seem to activate a very unusual substrate. Over-expression and purification of A domains from these four NRPS modules will be necessary to examine their substrate specificities. Substrate specificity of each A domain will therefore be determinated by analysis of the ATP-PPi exchange reaction with different substrate putatively incorporated into albicidin. Investigating albicidin backbone biosynthesis will be of great interest because such information adds to the limited knowledge as to how PKS and NRPS interact and how they might be manipulated to engineer novel molecules, and may explain how X. albilineans produces several structurally related, toxic compounds.
Cloning and sequencing of XALB2 showed that the same phosphopantetheinyl transferase is required for albicidin production in an X. albilineans strain from Florida and in an X. albilineans strain from Australia (Huang et al., 2000b), explaining the precedented results showing that strain LS156 mutated in xabA (100% identical to albX) was not complemented by pALB540, pALB571 and pALB639 (Rott et al., 1996). Mutant LS156 was shown to be complemented by a construction containing the coding sequence of xabA in fusion with lacZ, revealing that xabA is required for albicidin production and that no other cistron downstream from xabA was involved in albicidin production (Huang et al., 2000b). However, this complementation study did not allow determination of whether xabA is transcribed as a part of a larger operon. Here we disclose the complementation of mutant AM37 with a 2986 bp insert from X. albilineans containing albXXI (100% identical to xabA), confirming that albXXI is involved in albicidin biosynthesis and indicating that the promoter of albXXI is present in the 2986 bp insert and that albXXI is not expressed as part of a operon.
Cloning and sequencing of XALB3 showed that a heat shock protein HtpG was involved in albicidin production in X. albilineans. The heat shock protein HtpG is an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone. Hsp90 from eukaryotes has been demonstrated to possess chaperone activity (Jakob et al., 1995), acting as a non-ATP dependent “holder,” and it also has an important role in signal transduction and the cell cycle. This protein is essential in both drosophila and yeast (Borkovich et al., 1989; Cutforth and Rubin, 1994). In contrast, the HtpG gene can be deleted in E. coli with no effect on the viability of the strain with the exception of decreased growth rate at high temperatures (Bardwell and Craig, 1988). The in vivo role of the HtpG protein remains unknown. However, preliminary results indicated that HtpG facilitates de novo protein folding in stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE molecular chaperone system to interact with newly synthesized polypeptides (Thomas and Baneyx, 2000). Furthermore, HtpG was copurified in E. coli with MccB17 synthetase, an enzyme involved in the biosynthesis of the peptide antibiotic microcin B17 which inhibits DNA replication by induction of the SOS repair system, suggesting the requirement of HtpG for production of the antibiotic (Li et al., 1996). However, when microcin B17 production by the E. coli strain deleted for HtpG was compared to the one of the parental strain, there was no effect on microcin B17 production in vivo. This result implied that the copurification of HtpG with the MccB17 synthetase was potentially an artifact, or that another E. coli chaperone could substitute for HtpG (Milne et al., 1999). To examine the effect of HtpG on the reconstitution of MccB17 synthetase in vitro, the chaperone was expressed and purified as a fusion to a hexahistidine (His6) tag. Addition of the His6-HtpG did not stimulate MccB17 synthetase reconstitution or heterocyclisation activity in vitro, suggesting that HtpG mediates complex assembly or stabilizes protein subunits prior to the hetero-oligomerisation (Milne et al., 1999). Based on these results, we suggest that the function of AlbXXII is to mediate complex assembly by facilitating de novo protein folding of PKS and NRPS enzymes (AlbI, AlbIV, AlbVII and AlbIX) involved in the albicidin backbone biosynthesis.
Characterization of the complete sequence of XALB1, XALB2 and XALB3 clusters enables one to characterize all enzymes of the albicidin biosynthesis pathway including structural, resistance, secretory and regulatory elements, and to engineer overproduction of albicidin. For example one may insert expression enhancing DNA into the genome of X. albilineans in a position operable to enhance expression of the Albicidins Biosynthesis Gene Clusters. One may also modify naturally occurring Albicidins to obtain additional non-naturally occurring antibiotics by adding DNA encoding additional enzymes selected to produce a modified albicidin like molecule. This approach will allow (I) the purification of albicidin and the other compounds structurally related and potentially produced by the same biosynthesis apparatus; (ii) the characterization of chemical structure of albicidin; (iii) the investigation of mode of action of albicidin in the pathogenesis of X. albilineans in sugarcane; and (iv) the characterization of the bactericidal activity of albicidin. For example one may also increase the resistance of plants to damage from X. albilineans infection by inserting one or more of the resistance genes identified herein into the genome of the plant. One may also provide materials to prevent damage by albicidin produced by X. albilineans by applying an agent that blocks expression of the Albicidin Biosynthesis Gene Clusters to the plant to be protected. One may also use portions of the DNA of the Albicidin Biosynthesis Gene Clusters to obtain agents useful in blocking expression of albicidin by screening materials against a modified hast cell line that expresses the Albicidin Biosynthesis Gene Clusters and selecting for materials that stop or decrease albicidin production.







TABLE 1







Bacterial strains and plasmids used in this study









Strains
Relevant characteristicsa
Reference or source





E. coli




DH5α
F-/80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1
Gibco-BRL



hsdR17(rk−mk+) supE44 thi-1 gyrA96 relA1


DH5α MCR
DH5αmcrA mcrBC mrr
′′


Xcv 91-11B
Wild type strain of Xanthomonas axonopodis pv. vesicatoria
Astua-Monge et al.,



from tomato (race 3)
2000


Xcv 91-11BR1
Spontaneous Rifr derivative of Xcv 91-11B
This study


DH5αKT
Escherichia coli DH5α strain transformed by both pUFR043
′′



and pLAFR3 plasmids


DH5αAlbr
Spontaneous Albr derivative of DH5α
′′


DH5αAlbrKT
DH5αAlbr transformed by both pUFR043 and pLAFR3
′′



plasmids


Plasmids


PBR325
Tcr, Apr, Cmr
Gibco-BRL


pBCKS (+)
Cmr
Stratagene


pBluescript II KS (+)
Apr
′′


PRK2073
PRK2013 derivative, Kms (npt::Tn7), Spr, Tra+, helper plasmid
Leong et al., 1982


pUFR043
IncW Mob+ LacZα Gmr, Kmr, Cos
De Feyter and




Gabriel, 1991


pAlb540
47 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
Rott et al., 1996


pAlb571
36.8 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
′′


PAlb639
36 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
′′


pAM15.1
24 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM15 in pBR325, Kmr, Tcr, Apr, Cmr


pAM40.2
11 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM40 in pBR325, Kmr, Tcr, Apr, Cmr


pAM45.1
12 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM45 in pBR325, Kmr, Tcr, Apr, Cmr


pAM12.1
13 kb EcoR I fragment carrying Tn5 and flanking sequences of
A



mutant AM12 in pBR325, Kmr, Tcr, Apr, Cmr


PAM36.2
9 kb EcoR I fragment carrying Tn5 and flanking sequences of
A



mutant AM36 in pBR325, Kmr, Tcr, Apr, Cmr


pAlb389
37 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
This study


pAC389.1
2.9 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
′′


PAlb639A
9.4 kb insert from Xa23R1 in pUFR043, Gmr, Kmr
′′


PEV639
2.6 kb Sal I insert from Xa23R1 in pUFR043, Gmr, Kmr
′′


pBC/A′
7.5 kb Kpn I fragment carrying a part of fragment A from
′′



pAlb571 in pBCKS (+), Cmr


pBC/AF
15.2 kb EcoR I fragment carrying fragments A and F from
′′



pALB540 in pBCKS (+), Cmr


pBC/B
11.0 kb Kpn I fragment B from pAlb571 in pBCKS (+), Cmr
′′


pBC/C
6.0 kb Kpn I fragment C from pAlb571 in pBCKS (+), Cmr
′′


pBC/E
2.8 kb Kpn I fragment E from pAlb571 in pBCKS (+), Cmr
′′


pBC/F
2.5 kb Kpn I-EdoR I fragment F from pAlb571 in pBCKS (+),
′′



Cmr


pBC/G
1.9 kb EcoR I fragment G from pAlb571 in pBCKS (+), Cmr
′′


pBC/I
1.4 kb Kpn I-EcoR I fragment I from pAlb571 in pBCKS (+),
′′



Cmr


pBC/J
0.6 kb EcoR I fragment J from pALB540 in pBCKS (+), Cmr
′′


pBC/K
4.7 kb EcoR I fragment K from pALB540 in pBCKS (+), Cmr
′′


pBC/L
0.4 kb EcoR I fragment L from pALB540 in pBCKS (+), Cmr
′′


pBC/N
7.7 kb EcoR I fragment N from pALB540 in pBCKS (+), Cmr
′′


pUFR043/D=
2.2 kb EcoR IBSau3A I fragment carrying a part of fragment D
′′



from pAlb571 in pUFR043


pAM1
5 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM1 in pBluescript II KS (+), Kmr, Apr


pAM4
12 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM4 in pBluescript II KS (+), Kmr, Apr


pAM7
6 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM7 in pBluescript II KS (+), Kmr, Apr 


pAM10
7 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM10 in pBluescript II KS (+), Kmr, Apr


pAM29
10 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM29 in pBluescript II KS (+), Kmr, Apr


pAM37
6 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM37 in pBR325, Kmr, Tcr, Apr, Cmr


pAM52
5 kb EcoR I fragment carrying Tn5 and flanking sequences of
′′



mutant AM52 in pBluescript II KS (+), Kmr, Apr


PLAFR3
IncP, Mob+, LacZα, Tcr, cos
Staskawicz et al.,




1987


PLAFR3XhoI
pLAFR3 with a XhoI site added to the BamHI site using an
This study



adaptator


pBC/Op4Δ
BamHI-PstI fragment from pALB540 cloned between BamHI
′′



and PstI sites of pBCKS(+)


pBC/Op4ΔXhoI
PBC/Op4Δ with a XhoI site created by directed mutagenesis
′′



upstream from the BfrI site


pBC/Op4Δ/XALB2
EcoRI DNA fragment from pAC389.1 cloned into the EcoRI
′′



site of pBC/Op4ΔXhoI


pBC/Op3-4/XALB2
BfrI DNA fragment from pALB540 cloned into the BfrI site of
′′



pBC/Op4Δ/XALB2


pBKS/XALB3
SalI DNA fragment from pEV639 cloned into the SalI site of
′′



pBluescript II KS (+)


pBKS/XALB3XhoI
pBKS/XALB3 with a KhoI site created by directed mutagenesis
′′



to substitute the SalI site located on the KpnI side of the



polylinker


pBKS/Op3-4/
XhoI DNA fragment from pBC/Op3-4Δ/XALB2 cloned into the
′′


XALB2-3
SalI site of pBKS/XALB3XhoI


pOp3-4/XALB2-3
XhoI DNA fragment from pBKS/Op3-4/XALB2-3 cloned into
′′



the XhoI site of pLAFR3XhoI


pEValbXXII
albXXII in fusion with LacZ in pUFR043, Gmr, Kmr
′′


pEVHtpG
E. coli htpG in fusion with LacZ in pUFR043, Gmr, Kmr
′′


PGemT
ColE1 replicon, Apr, LacZα, single 3′-T overhangs at the
Promega



insertion site


PGemT/albXXII
PCR fragment containing albXXII cloned into pGemT
This study


PGemT/albXXII bis
BglII-SalIDNA fragment from pBKS/XALB3 cloned between
′′



the BglII and SalI sites of pGemT/albXXII


PGemT/HtpG
PCR fragment containing the E. coli htpG gene cloned into
′′



pGemT


DNA Fragment


PR37
1.1 kb Hind III-Hind III from pAM37
′′





aApr, Cmr, Gmr, Kmr, Rifr, Spr, Tcr: resistant to ampicilin, chloramphenicol, gentamycin, kanamycin, rifampicin, spectinomycin, tetracycline, respectively.


Tox-, deficient in albicidin production.


Tn5-gusA, Tn5-uidAl Kmr Tcr, forms transcriptional fusions.


Albr, Apr, Gmr, Rifrand Tcr: resistant to albicidin, ampicilin, gentamycin, rifampicin and tetracycline, respectively.













TABLE 2







Analysis of putative translational signals and location of all putative orfs identified in the


XALB1 gene cluster











Intergenic spacing between

Potential RBSa




consecutive ORFs in each

(distance from start
Start codon
Stop codon


putative operon
ORF
codon)
(position)
(position)





Operon 1 (strand +)







albI
GAGGG (5 b)
TTG (30166)
TAG (50805)


 45 b
albII
GAGGG (5 b)
ATG (50851)
TAA (51882)


ATG overlaps TAA
albIII
GAGGG (7 b)
ATG (51882)
TGA (52385)


GTG overlaps TGA
albIV
GAGG (7 b)
GTG (52382)
TAA (55207)


Operon 2 (strand −)



albV
GGAGG (8 b)
ATG (29929)
TAA (29210)


 87 b
aIbVI
AAGG (4 b)
GTG (29122)
TGA (28262)


 61 b
albVII
GAG (4 b)
ATG (28200)
TAG (25903)


 7 b
albVIII
AGGTG (4 b)
ATG (25895)
TAA (24903)


 20 b
albIX
GGTG (3 b)
ATG (24882)
TGA (19003)


Operon 3 (strand −)



albX
GGGGG (8 b)
ATG (14497)
TGA (14246)


 81 b
albXI
AGGAAA (6 b)
ATG (14164)
TGA (13217)


 5 b
albXII
GGCCTGA (5 b)
ATG (13211)
TAA (11856)


 36 b
albXIII
GGGG (3 b)
ATG (11819)
TAA (10866)


 12 b
albXIV
GGAG (8 b)
ATG (10853)
TAG (9363)


 41 b
albXV
GGAA (6 b)
ATG (9321)
TAG (7567)


208 b
albXVI
GGAGG (4 b)
ATG (7358)
TAG (7092)


Operon 4 (strand +)



albXVII
GGGAGG (5 b)
TTG (14909)
TGA (17059)


274 b
albXVIII
GCTCAG (8 b)
ATG (17334)
TGA (17747)


Overlap (17 b)
albXIX
AGG (9 b)
ATG (17728)
TGA (18330)


 41 b
albXX
GCAA (8 b)
ATG (18372)
TAG 18980)





aRibosomal Binding Site













TABLE 3







Deduced functions of the ORFs in the major albicidin biosynthetic


cluster X-ALB1











Number of




ORF
amino acids
Sequence homologa
Proposed functionb, c













Operon 1





AlbI
6879
XabB (AAK15074)
Polyketide — peptide synthase










PKS modules
PKS domains



PKS-1
AL  ACP1



PKS-2
KS1 KR ACP2 ACP3



PKS-3
KS2 PCP1



NRPS modules
NRPS domains



NRPS-1
C A PCP2



NRPS-2
C A PCP3



NRPS-3
C A PCP4



NRPS-4
C










Alibi
343
XabC (AAK15075)
C-methyltransferase


AlbIII
167
ComAB (CAA71583)
Activator of alb genes transcription


AlbIV
941
MycA (T44806)
Peptide synthase











WbpG (E83253)
NRPS module
NRPS domains




NRPS-5
      A PCP5










Operon 2





AlbV
239
Thp (AAK15074)
No function (transposition)


AlbVI
286
TcmP (AAA67510)
O-methyltransferase


AlbVII
765
HbaA (A58538)
4-hydroxybenzoate CoA ligase


albVIII
330
SyrP (AAB63253)
Regulation


AlbIX
1959
DhbF (CAB04779)
Peptide synthase










NRPS modules
NRPS domains



NRPS-6
      A PCP6



NRPS-7
C A PCP7










Operon 3





AlbX
83
MbtH (O05821)
Unknown


AlbXI
315
SyrC (U25130)
Thioesterase


AlbXII
451
BoxB (AAK006000.1)
Unknown


albXIII
317
hpd (AAK25001)
Esterase


albXIV
496
ActII-2 (p46105)
Albicidin transporter


AlbXV
584
hpd (08390)
Carbamoyl transferase


AlbXVI
88
OrfA (AAC03166)
No function (transposition)


Operon 4


albXVII
716
PabAB (CAC22117)
Para-amino benzoate synthase


Operon 5


albXVIII
137
ADCL (AAG06352)
No function (not functional)


albXIX
200
McbG (P05530)
Immunity against albicidin


albXX
202
UbiC (S25660)
4-hydroxybenzoate synthetase





aProtein accession numbers in Genbank are given in parentheses.


bNRPS and PKS domains are abbreviated as follows: A, adenylation;


ACP, acyl carrier protein;


AL, acyl CoA ligase;


C, condensation;


KR, ketoreductase;


KS, ketoacyl synthase;


PCP, peptidyl carrier protein.


cUnderlined domains are likely inactive due to the lack of highly conserved motifs.


dhypothetical protein













TABLE 4







Summary of results obtained from BLAST analyses.
















Putative
No. of










Alb
aa


Genbank


protein
residues
Protein homolog
Origin
accession #
Score
Expect
Identities
Positives
Gaps





AlbI
6879










PKS-1

XabB (4801 aa)
Xanthomonas albilineans
AAK15074
1352 bits (3498)
0.0
730/730
730/730
—









(100%)
(100%)




SafB (1770 aa)
Myxococcus xanthus
AAC44128
231 bits (589)
2e-59
175/532
269/532
23/532 (4%)









(32%)
(49%)


PKS-2

XabB (4801 aa)
X. albilineans
AAK15074
3464 bits (8983)
0.0
1882/1882
1882/1882
—









(100%)
(100%)




PksM (4273 aa)
Bacillus subtilis
CAB13603
887 bits (2292)
0.0
626/1896
938/1896
140/1896 =









(33%)
(49%)
7%


PKS-3

XabB (4801 aa)
X. albilineans
AAK15074
1274 bits (3296)
0.0
653/653
653/653
—









(100%)
(100%)




PksM (4273 aa)
B. subtilis
CAB13603
577 bits (1486)
e-163
293/584
391/584
17/584 (2%)









(50%)
(66%)


NRPS-1

XabB (4801 aa)
X. albilineans
AAK15074
1934 bits (5010)
0.0
1035/1046
1039/1046
—









(99%)
(99%)




NosA (4379 aa)
Nostoc sp.
AF204805
618 bits (1594)
e-176
398/1104
586/1104
86/1104









(36%)
(53%)
(7%)


NRPS-2

NosA (4379 aa)
Nostoc sp.
AF204805
416 bits (1069)
e-115
337/1127
496/1127
128/1127









(29%)
(43%)
(11%)




Peptide synthase
Anabaena sp.
CAC01604
402 bits (1034)
e-111
315/1073
479/1073
114/1073




(5060 aa)




(29%)
(44%)
(10%)


NRPS-3

XabB (4801 aa)
X. albilineans
AAK15074
1847 bits (4784)
0.0
997/1044
1007/1044
—









(95%)
(96%)




NosA (4379 aa)
Nostoc sp.
AF204805
610 bits (1573)
e-173
392/1069
571/1069
86/1069









(36%)
(52%)
(8%)


NRPS-4

XabB (4801 aa)
X. albilineans
AAK15074
889 bits (2297)
0.0
468/468
468/468
—









(100%)
(100%)




NosC (3317 aa)
Nostoc sp.
AAF17280
240 bits (613)
2e-62
156/438
229/438
20/438 (4%)









(35%)
(51%)


AlbII
343
XabC (343 aa)
X. albilineans
AAK15075
633 bits (1633)
0.0
343/343
343/343
—









(100%)
(100%)




MtmMII (326 aa)
Streptomyces argillaceus
AAD55584
144 bits (361)
1e-34
98/323
154/323
4/323 (1%)









(30%)
(47%)




TcmO (339 aa)
S. glaucescens
P39896
81.7 bits (199)
1e-14
79/314
140/314
12/314 (3%)









(25%)
(44%)


AlbIII
167
comA operon
E. coli
AAC74756
133 bits (335)
1e-30
68/135
89/135
—




protein




(50%)
(65%)




2 (136 aa)




ComAB (116 aa)
Bacillus licheniformis
CAA71583
97.6 bits (242)
8e-20
53/111
68/111
1/111 (0%)









(47%)
(60%)


AlbIV
941


PKS-4

BA3 (6359 aa)
B. licheniformis
AAC06348
361 bits (926)
2e-98
190/441
267/441
14/441 (3%)









(43%)
(60%)




WbpG (377 aa)
Pseudomonas aeruginosa
E83253
81.6 bits (200)
4e-15
44/119
70/119
4/119 (3%)









(36%)
(57%)


AlbV
239
Thp (240 aa)
X. albilineans
nd
nd
0.0
240/240
240/240
—




IS transposase




(100%)
(100%)




(260 aa)
Yersinia pestis
AAC82714
160 bits (404)
1e-38
87/183
122/183
2/183 (1%)









(47%)
(66%)


AlbVI
286
Hypothetical protein
Mycobacterium
AAK46042
138 bits (347)
6e-32
92/224
125/224
18/224 (8%)




TcmP (276 aa)
tuberculosis



(41%)
(55%)





Pasteurella multocida
AAK03406
36.6 bits (83)
0.24
32/132
65/132
29/197 (6%)









(28%)
(49%)


AlbVII
765
4-hydroxybenzoate-
Rhodopseudomonas
AAA62604
203 bits (513)
5e-51
156/492
242/492
31/492 (6%)




CoA ligase (539 aa)
palustris



(31%)
(48%)


AlbVIII
330
SyrP Like (339 aa)
S. verticillus
AF210249
245 bits (619)
6e-64
130/309
182/309
2/309 (0%)









(42%)
(58%)




SyrP (353 aa)
Pseudomonas syringae
AAB63253
182 bits (458)
5e-45
106/306
155/306
4/306 (1%)









(34%)
(50%)


AlbIX
1959 


NRPS-6

XabB (4801 aa)
X. albilineans
AAK15074
481 bits (1239)
e-135
286/608
374/608
23/208 (3%)









(47%)
(61%)




DhbF (1278 aa)
B. subtilis
CAB15186
354 bits (908)
1e-96
222/608
341/608
21/608 (3%)









(36%)
(55%)


NRPS-7

XabB (4801 aa)
X. albilineans
AAK15074
874 bits (2258)
0.0
515/1110
682/1110
52/1110









(46%)
(61%)
(4%)




NosA (4379 aa)
Nostoc sp.
AF204805
551 bits (1420)
e-155
388/1148
583/1148
84/1148









(33%)
(49%)
(7%)


AlbX
83
Hypothetical protein
P. aeruginosa
AAG05800
75.6 bits (185)
1e-13
34/61
44/61
—




(72 aa)




(55%)
(71%)




MbtH (71 aa)
M. tuberculosis
CAB08480
59 bits (142)
9e-09
25/55
37/55
—









(45%)
(66%)


AlbXI
315
SyrC (433 aa)
P. syringae
AAA85161
34.4 bits (78)
1.9
23/93
40/93
—









(24%)
(42%)




Hydrolase (261 aa)
S. coelicolor
CAA16200
34 bits (77)
2.9
19/60
30/60
—









(31%)
(49%)


AlbXII
451
BoxB (473 aa
Azoarcus evansii
AAK00599
293 bits (751)
3e-78
174/448
243/448
12/448 (2%)









(38%)
(53%)


AlbXIII
317
Hypothetical protein
Caulobacter crescentus
AAK25001
99.5 bits (247)
5e-200
88/296
125/296
5/296 (1%)




(335 aa)




(29%)
(41%)




Plasma PAF
Canis familiaris
AAC48484
37.5 bits (86)

43/156
56/156
44/156




acetylhydrolase




(28%)




(444 aa)


AlbXIV
496
Putative trans-
S. coelicolor
CAB90983
225 bits (574)
0
154/465
240/465
8/465 (1%)




membrane efflux




(33%)
(51%)




protein (505 aa)




AlbF, putative
X. albilineans
AF403709
736 bits (1900)

496/496
496/496
—




albicidin efflux




(100%)
(100%)




pump (496 aa)


AlbXV
584
Probable carbamoyl
P. aeruginosa
AAG08390
201 bits (513)
1e-50
158/458
222/458
39/458 (8%)




transferase (585 aa)




(34%)
(47%)




BlmD (545 aa)
S. verticillus
AAG02370
192 bits (506)
1e-47
149/441
209/441
33/441 (7%)









(33%)
(46%)


AlbXVI
88
Transposase
X. axonopodis
AF263433
64.8 bits (157)
2e-10
27/45
40/45
—




(363 aa)




(60%)
(88%)




Transposase OrfA
Desulfovibrio vulgaris
AAC03166
61.0 bits (147)
3e-09
29/54
38/54
—




(88 aa)




(53%)
(69%)


AlbXVII
716
Para-aminobenzoate
Streptomyces griseus
CAC22117
503 bits (1295)
e-141
302/699
409/699
36/699 (5%)




synthase (723 aa)




(43%)
(58%)


AlbXVIII
137
4-amino-4-
P. aeruginosa
AAG06352
81.4 bits (200)
4e-15
46/105
65/105
—




deoxychorismate




(43%)
(61%)




lyase (271 aa)


AlbXIX
200
McbG (187 aa)
E. coli
CAA30724
60.5 bits (145)
9e-09
36/141
58/141
5/141 (3%)









(25%)
(40%)


AlbXX
202
4-hydroxybenzoate
E. coli
AAC77009
45.6 bits (107)
5e-04
42/161
21/161
—




synthase (202 aa)




(26%)
(13%)


AlbXXI
278
XabA (278 aa)
X. albilineans
AAG28384
430 bits (1106)
0
278/278
278/278
—









(100%)
(100%)


AlbXXII
634
Heat shock protein
P. aeruginosa
AAG04985
1051 bits (2688)
0
523/634
588/634
—




HtpG (634)




(82%)
(92%)




Heat shock protein
E. coli
AAC73575
743 bits (1899)
0
376/624
476/624
4/624 (0%)




HtpG (624)




(60%)
(76%)

















TABLE 5







Comparison of conserved sequences in C domains



of peptide synthestases and in putative C


domains of the Alb modules












Sequences






conserved in





peptide




Core
synthetases*
Sequence
Alb module





C1
SxAQxR(L/M)
TYAQERLWLV
NRPS-1




(W/Y)xL


 




STAQERMWFL
NRPS-2


 




SYAQERLWLV
NRPS-3


 




SLFQERLWFV
NRPS-4


 




SYQQERLWFV
NRPS-7


 


C2
RHExLRTxF
RHEVLRTRF
NRPS-1 and NRPS-3


 




RHAVLRTHF
NRPS-2


 




RHEILRTRF
NRPS-4


 




RHETLRTRI
NRPS-7


 


C3
MHHxISDG(W/V)S
IHHIISDGWS
NRPS-1 and NRPS-3


 




IHHIVFDGWS
NRPS-2


 




MHHLIYDAWS
NRPS-4


 




MHHIICDGWS
NRPS-7


 


C4
YxD(F/Y)AVW
YADYALW
NRPS-1 and NRPS-3


 




YADYARW
NRPS-2


 




YADYAIW
NRPS-4


 




YADYATW
NRPS-7


 


C5
(I/V)GxFVNT(Q/L)
IGFFINILPLR
NRPS-1, NRPS-3 and



(C/A)xR

NRPS-4


 




IGLFVNTLAVR
NRPS-2


 




IGFFVNILAVR
NRPS-7


 


C6
(H/N)QD(Y/V)PFE
HQSVPFE
NRPS-1 and NRPS-3


 




HQDVPFE
NRPS-2


 




NQALPFE
NRPS-4


 




HRALPFE
NRPS-7


 


C7
RDxSRNPL
RDSSQIPL
NRPS-1 and NRPS-3


 




RDTARNPL
NRPS-2


 




RDTSRIPL
NRPS-4


 




RDSSQIPL
NRPS-7





*Sourced from Marahiel et al., 1997














TABLE 6







Comparison of conserved sequences in A domains of peptide



synthestases and in putative A domains of the Alb modules












Sequences conserved in





Core
peptide synthetases*
Sequence
Alb module





A1
L(T/S)YxEL
WSYAQL
NRPS-1 and NRPS-3



 




LSYAQL
NRPS-2


 




MSYGQL
NRPS-5


 




FSYRQL
PKS-4


 




LSYAQL
NRPS-6 and NRPS-7


 


A2
LKAGxAYL(V/L)P(L/I)D
FKAGACYVPID
NRPS-1 and NRPS-3


 




SLCGAASVLID
NRPS-2


 




MKAGAAYVPID
NRPS-5


 




LAGGLVFAPIN
PKS-4


 




LKAGGCYVPLD
NRPS-6 and NRPS-7


 


A3
LAYxxYTSG(S/T)TGxPKG
LACVMVTSGSTGRPKG
NRPS-1 and NRPS-3


 




?TRTIMVESGSLSSRLL?
NRPS-2


 




PVYCIYTSGSTGSPKG
NRPS-5


 




PAVMICTSGSTGTPKA
PKS-4


 




LAYVMYTSGSTGRPKG
NRPS-6 et NRPS-7


 


A4
FDxS
FAVS
NRPS-1 and NRPS-3


 




FDAA
NRPS-2


 




FDLT
NRPS-5


 




FAYG
PKS-4


 




FAIS
NRPS-6 and NRPS-7


 


A5
NxYGPTE
NNYGCTE
NRPS-1 and NRPS-3


 




?AAYGNAE?
NRPS-2


 




NEYGPTE
NRPS-5


 




DGIGCTE
PKS-4


 




YIYGCTE
NRPS-6 and NRPS-7


 


A6
GELxIxGxG(V/L)ARGYL
GELHVHSVGMARGYW
NRPS-1 and NRPS-3


 




np
NRPS-2


 




GQIHIGGAGVAIGYV
NRPS-5


 




GSLWVRGNTLTRGYV
PKS-4


 




GEVHIESLGITHGYW
NRPS-6 and NRPS-7


 


A7
Y(R/K)TGDL
YKTGDM
NRPS-1 and NRPS-3


 




?YKTDAL?
NRPS-2


 




YASGDL
NRPS-5


 




?FDTRDL?
PKS-4


 




YRTGDM
NRPS-6 and NRPS-7


 


A8
GRxDxQVKIRGxRIELGEIE
GRQDFEVKVRGHRVDTRQVE
NRPS-1 and NRPS-3


 




?GSLDVQSRIDDPRIDLCVVE?
NRPS-2


 




GRKDSQIKLRGYRIELGEIE
NRPS-5


 




?GRMGSAIKINGCWLSPETLE?
PKS-4


 




GRRDYEVKVRGYRVDVRQVE
NRPS-6 and NRPS -7


 


A9
LPxYM(I/V)P
LPTYMLP
NRPS-1 and NRPS-3


 




?LPDYLLP?
NRPS-2


 




LPEYMLP
NRPS-5


 




?LGKHHYP?
PKS-4


 




LPTYMLP
NRPS-6 and NRPS-7


 


A10
NGK(V/L)DR
NGKLDR
NRPS-1 and NRPS-3


 




?HGRVDL?
NRPS-2


 




NGKVNR
NRPS-5


 




?SGKVIR?
PKS-4


 




NGKLDT
NRPS-6 and NRPS-7





*Sourced from Marahiel et al., 1997


?non conserved sequences


np: not present














TABLE 7







Comparison of conserved sequences in PCP and TE



domains of peptide synthestases and in putative


PCP and TE domains of the Alb modules












Sequences conserved





Do-
in peptide

Alb module


main
synthetases*
Sequence
(domain)





PCP
DxFFxxLGG(H/D)S(L/I)
D-FFAVGGHSVL
PKS-3






(PCP1)


 




DNFFALGGHSLS
NRPS-1 and





NRPS-3





(PCP2 and





PCP4)


 




DNFFELGGHSVL
NRPS-2





(PCP3)


 




DNFFELGGHSLS
NRPS-5





(PCP5)


 




DNFFNLGGHSLL
NRPS-6 and





NRPS-7





(PCP6 and





PCP7)


 


TE
G(H/Y)SxG
GWSSG
NRPS-7





*Sourced from Marahiel et al., 1997
















TABLE 8









Position in GsrA (Phe) and




variability



















235
236
239
278
299
301
322
330
331
517



Domains
0
+/−
++
++
++
+/−
++
+/−
++
0





Alb NRPS-1
A
V
K
Y
V
A
N
D
A
K



 


Alb NRPS-3
A
V
K
Y
V
A
N
D
A
K


 


TyrB-M1 (Pro)
D
V
Q
S
I
A
N
V
V
K


 


VirS (Pro)
D
V
Q
Y
A
A
H
V
M
K


 


HVCL
G
A
L
H
V
V
G
S
I
K


 


Alb NRPS-6
A
I
K
Y
F
S
I
D
M
K


 


Alb NRPS-7
A
I
K
Y
F
S
I
D
M
K


 


VirS (Pro)
D
V
Q
Y
A
A
H
V
M
K


 


EntF-M1 (Ser)
D
V
W
H
F
S
L
V
D
K


 


β-Ala code
V
D
W
V
I
S
L
A
D
K


 


Alb NRPS-5
D
L
T
K
I
G
E
V
G
K


 


BacC-M5 (Asn)
D
L
T
K
I
G
E
V
G
K


 


TyrC-M1 (Asn)
D
L
T
K
I
G
E
V
G
K


 


Asn code
D
L
T
K
L
G
E
V
G
K
















TABLE 9







Complementation studies of Xa23RI insertion mutants









Recipient












Donor
AM12
AM13
AM36
AM10
AM15





pEV639
+
+
+
−
−


pEValbXXII
+
+
+
−
−


pEVHtpG
+
+
+
−
−


pALB639
+
+
+
−
−


pUFR043
−
−
−
−
−


none
−
−
−
−
−





+: restoration of albicidin production by alb− mutant,


−: no complementation.


All experiments were performed at least in duplicate with at least 2 exconjugants obtained from two independent triparental conjugations.













TABLE 10







Albicidin production assays with X. axonopodis pv. vesicatoria


exconjugants harbouring different plasmids: analysis of growth


inhibition of E. coli DH5αKT (susceptible to albicidin) and


DH5αAlbrKT (resistant to albicidin) in assays performed


with different antibiotic combinations (no antibiotic, tetracycline


only, kanamycin only and tetracycline + kanamycin).









Combination of plasmids














pUFR043
pUFR043
PALB571
PALB571


Bioassay medium
Tester
and
and
and
and


containing
strain
pLAFR3
pOp3-4/XALB2-3
pLAFR3
pOp3-4/XALB2-3





No antibiotic
DH5αKT
−
−
−
−



DH5αAlbrKT
−
−
−
−


Tetracycline
DH5αKT
−
−
−
+



DHSαAlbrKT
−
−
−
−


Kanamycin
DH5αKT
+
+
+
+



DH5αAlbrKT
+
+
+
+


Tetracycline + kanamycin
DH5αKT
+
+
+
+



DH5αAlbrKT
+
+
+
+





+: presence of a growth inhibition zone


All experiments were performed at least in duplicate with at least 2 exconjugants obtained from two independent triparental conjugations.






REFERENCES


    1. Albertini, A. M., Caramori, T., Scoffone, F., Scotti, C. & Galizzi, A. (1995). Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb. Microbiology 141, 299-309.
    2. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein data base search programs,” Nucleic Acids Res. 25, 3389-3402.
    3. Aparicio, J. F., Molnar, I., Schwecke, T., Konig, A., Haydock, S. F., Khaw, L. E., Staunton, J. & Leadlay, P. F. (1996). Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169, 9-16.
    4. Astua-Monge, G., Minsavage, G. V., Stall, R. E., Davis, M. J., Bonas, U. and Jones, J. B. (2000). Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. vesicatoria is specified by a plant-inducible avirulence gene. Mol. Plant Microbe Interact. 13:911-21.
    5. August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T. W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X., Hutchinson, C. R. & Floss, H. G. (1998). Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol. 5, 69-79.
    6. Bardwell, J. C. & Craig, E. A. (1988). Ancient heat shock gene is dispensable. J. Bacteriol. 170, 2977-83.
    7. Birch, R. G. (2001). Xanthomonas albilineans and the antipathogenesis approach to disease control. Mol. Plant Pathol. 2, 1-11.
    8. Birch, R. G. & Patil, S. S. (1983). The relation of blocked chloroplast differentiation to sugarcane leaf sca1 disease. Phytopathology 73, 1368-1374.
    9. Birch, R. G. & Patil, S. S. (1985a). June 1985. Antibiotic and process for the production thereof U.S. Pat. No. 4,525,354.
    10. Birch, R. G. & Patil, S. S. (1985b) Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli. J. Gen. Microbiol. 131, 1069-1075.
    11. Birch, R. G. & Patil, S. S. (1987a). Correlation between albicidin production and chlorosis induction by Xanthomonas albilineans, the sugarcane leaf scald pathogen. Physiol. Mol. Plant Pathol. 30, 199-206.
    12. Birch, R. G. & Patil, S. S. (1987b). Evidence that an albicidin-like phytotoxin induces chlorosis in sugarcane leaf scald disease by blocking plastid DNA replication. Mol. Plant Pathol. 30, 207-214.
    13. Borkovich, K. A., Farrelly, F. W., Finkelstein, D. B., Taulien, J. & Lindquist, S. (1989). Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9, 3919-30.
    14. Brendel, V. & Trifonov, E. N. (1984). A computer algorithm for testing potential prokaryotic terminators. Nucl. Acids Res. 12 4411-4427.
    15. Cane, D. E. (1997). A special thematic issue on polyketide and nonribosomal polypeptide biosynthesis. Chem. Rev. 97, 2463-2706.
    16. Cane, D. E., Walsh, C. T. & Khosla, C. (1998). Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 282, 63-8.
    17. Cane, D. E. & Walsh, C. T. (1999). The parallel and convergent universes of polyketide synthases and nonribosomal peptide synthetases. Chem. Biol. 6, R319-R325.
    18. Challis, G. L., Ravel, J. & Townsend, C. A. (2000). Predictive, structure-based model of amino acid recognition by nonribosomal peotide synthetase adenylation domains. Chem. Biol. 7, 211-224.
    19. Conti, E., Stachelhaus, T. Marahiel, M. A. & Brick, P. (1997). Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174-4183.
    20. Criado, L. M., Martin, J. F. & Gil, J. A. (1993). The pab gene of Streptomyces griseus, encoding p-aminobenzoic acid synthase, is located between genes possibly involved in candicidin biosynthesis. Gene 126, 135-139.
    21. Cutforth, T. & Rubin, G. M. (1994). Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila. Cell. 77, 1027-36.
    22. De Feyter, R. & Gabriel, D. W. (1991). Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum. J. Bacteriol. 173, 336-342.
    23. von Döhren, H., Dieckmann, R. & Pavela-Vrancic, M. (1999). The nonribosomal code. Chem. Biol. 6, R273-R279.
    24. Du, L., Sanchez, C., Chen, M., Edwards, D. J. & Shen, B. (2000). The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonrobosomal peptide synthetases and a polyketide synthase. Chem. Biol. 7, 623-642.
    25. Duitman, E. H., Hamoen, L. W., Rembold, M., Venema, G., Seitz, H., Saenger, W., Bernhard, F., Reinhardt, R., Schmidt, M., Ullrich, C., Stein, T., Leenders, F. & Vater, J. (1999). The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc. Natl. Acad. Sci. USA 96, 13294-13299.
    26. Gabriel, D. W & De Feyter, R. (1992). RFLP analyses and gene tagging for bacterial identification and taxonomy. In Molecular Plant Pathology: A Practical Approach (Gurr, S. J., McPherson, M. J. & Bowles, D. J. Ed.), vol 1, pp51-66, IRL Press, Oxford.
    27. Garrido, M. C., Herrero, M., Kolter, R. & Moreno, F. (1988). The export of the DNA replication inhibitor Microcin B17 provides immunity for the host cell. EMBO J. 7, 1853-1862.
    28. Gehring, A. M., DeMoll, E., Fetherston, J. D., Mori, I., Mayhew, G. F., Blattner, F. R., Walsh, C. T. & Perry, R. D. (1998). Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis. Chem. Biol. 5, 573-86.
    29. Guenzi, E., Galli, G., Grgurina, I., Gross, D. C. & Grandi, G. (1998). Characterization of the syringomycin synthetase gene cluster. A link between prokaryotic and eukaryotic peptide synthetases. J. Biol. Chem. 273, 32857-32863.
    30. Huang, G., Zhang, L. & Birch, R. G. (2000a). Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans. Gene 255, 327-333.
    31. Huang, G., Zhang, L. & Birch, R. G. (2000b). Albicidin antibiotic and phytotoxin biosynthesis in Xanthomonas albilineans requires a phosphopantetheinyl transferase gene. Gene 258, 193-199.
    32. Huang, G., Zhang, L. & Birch, R. G. (2001). A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans. Microbiology 147, 631-642.
    33. Jakob, U., Lilie, H., Meyer, I. & Buchner, J. (1995). Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J. Biol. Chem. 270, 7288-94.
    34. Konz, D., Klens, A., Schorgendorfer, K. & Marahiel, M. A. (1997). The bacitracin biosynthesis operon of Bacillus licheniformis ATCC10716: molecular characterization of three multi-modular peptide synthetase. Chem. Biol. 4, 927-937.
    35. Leong, S. A., Ditta, G. S. & Helinski, D. R. (1982). Heme biosynthesis in Rhizobium: identification of a cloned gene coding for aminolevulinic acid synthetase from Rhizobium meliloti. J Biol. Chem. 257, 8724-8730.
    36. Li, Y. M., Milne, J. C., Madison, L. L., Kolter, R. & Walsh, C. T. (1996). From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase. Science. 274, 1188-93.
    37. Liu, J., Duncan, K. & Walsh, C. T. (1989). Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthesis genes: identification of ent A and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate deshydrogenase. J. Bacteriol. 171, 791-798.
    38. Marahiel, M. A. Stachelhaus, T. & Mootz, H. D. (1997). Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651-2673.
    39. Mayorga, M. E. & Timberlake, W. E. (1992). The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases. Mol. Gen. Genet. 235, 205-212.
    40. McCarthy, J. E. & Gualerzi, C. (1990). Translational control of prokaryotic gene expression. Trends Genet. 6, 78-85.
    41. McDaniel, R., Thamchaipenet, A., Gustafsson, C., Fu, H., Betlach, M. & Ashley, G. (1999). Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products. Proc. Natl. Acad. Sci. USA 96, 1846-1851.
    42. Milne, J. C., Roy, R. S., Eliot, A. C., Kelleher, N. L., Wokhlu, A., Nickels, B. & Walsh, C. T. (1999). Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17. Biochemistry. 38, 4768-81.
    43. Mohamed, I. S., Rott, P., Davis, M. J. & Chatenet, M. (1996). Differentiation of Xanthomonas albilineans strains based on multiplication of the pathogen in sugarcane varieties. In Proceedings XXII Congress of the International Society of Sugarcane Technologists. (Cock J. H. and Brekelbaum T. Ed.), vol. 2, pp486-492, Cartagena De Indias, Colombia, 5-14 September 1995.
    44. Mohamed, M. E., Zaa, A., Ebenau-Jehle, C. & Fuchs, G. (2001). Reinvestigation of a new type of aerobic benzoate metabolism in the proteobacterium Azoarcus evansii. J. Bacteriol. 183, 1899-1908.
    45. Motamedi, H. & Shafiee, A. (1998). The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534.
    46. Normark, S., Bergström, S., Edlund, T., Grundström, T., Jaurin, G., Lindberg, F. P. & Olsson O. (1983). Overlapping genes. Annu. Rev. Genet. 17, 499-525.
    47. Paitan, Y., Orr, E., Ron, E. Z. & Rosenberg, E. (1999). Genetic and functional analysis of genes required for the post-modification of the polyketide antibiotic TA of Myxococcus xanthus. Microbiology 145, 3059-67.
    48. Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E. & Khosla C. (2001). Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790-1792.
    49. Quadri, L. E., Sello, J., Keating, T. A., Weinreb, P. H. & Walsh, C. T. (1998). Identification of a Mycobaterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem. Biol. 5, 631-645.
    50. Ricaud, C. & Ryan, C. C. (1989). Leaf scald. In Diseases of sugarcane: major diseases. (Ricaud, C. Egan, B. T., Gillaspie, Jr., A. G. & Hughes, C. G. ed.), pp. 39-58, Elsevier Science Publishers B.V., Amsterdam.
    51. Rodriguez, E. & McDaniel, R. (2001). Combinatorial biosynthesis of antimicrobials and other natural products. Curr. Opin. Microbiol. 4, 526-34.
    52. Rott, P. C., Costet, L., Davis, M. J., Frutos, R. & Gabriel D. W. (1996). At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans. J. Bacteriol. 178, 4590-4596.
    53. Rott, R. & Davis, M. J. (2000). Leaf scald. In A guide to sugarcane diseases. (Rott, P., Bailey, R. A., Comstock, J. C., Croft, B. J. and Saumtally, A. S. ed.), pp. 38, Cirad/Issct, Montpellier.
    54. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: A laboratory Manual (2nd edn), Cold Spring Harbor Lab. Press, Plainview, N.Y.
    55. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. USA 74, 5463-5467
    56. Siebert, M., Bechthold, A., Melzer, M., May, U., Berger, U. Schroder, G., Schroder, J., Severin, K. & Heide, L. (1992). Ubiquinone biosynthesis. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydrobenzoate octaprenyl transferase from Escherichia coli. FEBS Lett. 307, 347-350.
    57. Stachelhaus, T., Mootz, H. D. & Marahiel, M. A. (1999). The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493-505.
    58. Staskawicz, B. Dahlbeck, D., Keen, N. and Napoli, C. (1987). Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycenea. J. Bacteriol. 169: 5789-5794.
    59. Takano, Y., Kubo, Y., Shimizu, K., Mise, K., Okuno, T. & Furusawa, I. (1995). Structural analysis of PKS1, a polyketide synthase gene involved in melanin biosynthesis in Colletotrichum lagenarium. Mol. Gen. Genet. 249, 162-167.
    60. Tang, L., Yoon, Y. J., Choi, C. Y. & Hutchinson, C. R. (1998). Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei. Gene 216, 255-65.
    61. Thomas, J. G. & Baneyx, F. (2000). ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. Mol. Microbiol. 36, 1360-70.
    62. Valdez, F., Gonzalez-Ceron, G., Kieser, H. M., Servin-Gonzalez, L. (1999). The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator. Microbiology 145, 2365-74.
    63. Wall, M. K. & Birch, R. G. (1997). Genes for albicidin biosynthesis and resistance span at least 69 kb in the genome of Xanthomonas albilineans. Lett. Appl. Microbiol. 24,256-260.
    64. Yakimov, M. M., Kroger, A., Slepak, T. N., Giuliano, L., Timmis, K. N. & Golyshin, P. N. (1998). A putative lichenysin A synthase operon in Bacillus licheniformis: initial characterization. Biochim. Biophys. Acta. 1399, 141-153.
    65. Yazgan, A., Ozcengiz, G. & Marahiel M. A. (2001). Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin. Biochim. Biophys. Acta. 1518, 87-94.
    66. Yu, J. H. & Leonard, T. J. (1995). Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase. J. Bacteriol. 177, 4792-4800.
    67. Yu, S., Fiss, E., & Jacobs, W. R. (1998). Analysis of the exochelin locus in Mycobacterium smegmatis: biosynthesis genes have homology with genes of the peptide synthetase family. J. Bacteriol. 180, 4676-4685.
    68. Zhang, L. & Birch, R. G. (1997). The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane. Proc. Natl. Acad. Sci. USA 94, 9984-9989.
    69. Zhang, J. H., Quigley, N. B. & Gross, D. C. (1995). Analysis of the syrB and syrC genes of Pseudomonas syringae pv. syringae indicates that syringomycin is synthesized by a thiotemplate mechanism. J. Bacteriol. 177, 4009-4020.
    70. Zhang, J. H., Quigley, N. B. & Gross, D. C. (1997). Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 63, 2771-2778.
    71. Zhang, L., Xu, J. & Birch, R. G. (1998). Factors affecting biosynthesis by Xanthomonas albilineans of albicidins antibiotics and phytotoxins. J. Appl. Microbiol. 85, 1023-1028.
    72. Zhang L., Xu, J. & Birch, R. G. (1999). Engineered detoxification confers resistance against a pathogenic bacterium. Nat Biotechnol. 17:1021-1024.

## Claims

1. A transformed isolated host cell that comprises one or more genetic constructs that comprises SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3, wherein said transformed host cell produces albicidin.

2. The transformed isolated host cell of claim 1 wherein said genetic construct contains a combination of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

3. The transformed isolated host cell claim 1 wherein said host cell has been transformed with a first genetic construct comprising SEQ ID NO:1, a second genetic construct comprising SEQ ID NO:2 and a third genetic construct comprising SEQ ID NO:3.

4. A transformed isolated host cell comprising one or more polynucleotides encoding a biosynthetic pathway for albicidin production, said polynucleotides encoding SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 39, 40, 41, 42, 43, 44, 45, 46 and 47 and wherein said host cell produces albicidin.

5. A combination of isolated polynucleotides that encodes SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 39, 40, 41, 42, 43, 44, 45, 46 and 47.

6. The combination of isolated polynucleotides according to claim 5, wherein said combination of polynucleotides comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

7. A method of making an antibiotic comprising culturing a transformed host cell according to claim 4 under conditions that allow for the production of said antibiotic.

8. The method according to claim 7, further comprising the isolation of said antibiotic.