Functional and Transcriptional Analyses of a Fengycin Synthetase Gene, fenC, from Bacillus subtilis

A 37-kb DNA fragment containing five fengycin synthetase genes, including fenC, fenD, fenE,fenA, and fenB, was cloned and sequenced. Among these genes, fenC encodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activate l-glutamic acid and l-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon of fenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon of fenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription of fenC is positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis by B. subtilis F29-3.

Related papers:

title: Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3.
abstract: Fengycin is an antifungal lipopeptide complex produced by Bacillus subtilis strain F-29-3. It inhibits filamentous fungi but is ineffective against yeast and bacteria. The inhibition is antagonized by sterols, phospholipids and oleic acid, whereas two other unsaturated fatty acids increase the antifungal effect. Fengycin consists of two main components differing by one amino acid exchange. Fengycin A is composed of 1 D-Ala, 1 L-Ile, 1 L-Pro, 1 D-allo-Thr, 3 L-Glx, 1 D-Tyr, 1 L-Tyr, 1 D-Orn, whereas in fengycin B the D-Ala is replaced by D-Val. The lipid moiety of both analogs is more variable, as fatty acids have been identified as anteiso-pentadecanoic acid (ai-C15), iso-hexadecanoic acid (i-C16), n-hexadecanoic acid (n-C16), and there is evidence for further saturated and unsaturated residues up to C18.

title: Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis
abstract: A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.

title: Nucleotide sequence of 5' portion of srfA that contains the region required for competence establishment in Bacillus subtilus
abstract: The nucleotide sequence of the 20,535 base pairs of the 5' end of the srfA operon, containing the region required for competence development, was determined. This included the srfA promoter region, the first open reading frame, srfAA, encoding surfactin synthetase I and part of the second open reading frame, srfAB, encoding surfactin synthetase II. Three amino acid-activating domains characteristic of those found in peptide synthetases could be discerned in both srfAA (activating Glu, Leu and D-Leu) and srfAB (activating Val, Asp, and D-Leu). The presence of a conserved spacer motif in the amino-terminal end of srfAA suggests that the srfAA product may not initiate surfactin synthesis. The portion of srfA that contains the region required for competence is composed of srfAA and the first amino acid-activating domain of srfAB.

title: Nonribosomal biosynthesis of peptide antibiotics.
abstract: Peptide antibiotics are known to contain non-protein amino acids, D-amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N-methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the 'thiotemplate multienzymic mechanism'. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N-methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4'-phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine, cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and linear gramicidin are discussed. The structures of the multienzymes, their genetic organization, the biological functions of these peptides and results on related systems are discussed.

title: A nonribosomal system of peptide biosynthesis.
abstract: This review covers peptide structures originating from the concerted action of enzyme systems without the direct participation of nucleic acids. Biosynthesis proceeds by formation of linear peptidyl intermediates which may be enzymatically modified as well as transformed into specific cyclic structures. The respective enzyme systems are constructed of biosynthetic modules integrated into multienzyme structures. Genetic and DNA-sequence analysis of biosynthetic gene clusters have revealed extensive similarities between prokaryotic and eukaryotic systems, conserved principles of organisation, and a unique mechanism of transport of intermediates during elongation and modification steps involving 4'-phospho-pantetheine. These similarities permit the identification of peptide synthetases and related aminoacyl-ligases and acyl-ligases from sequence data. Similarities to other biosynthetic systems involved in the assembly of polyketide metabolites are discussed.

title: Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases
abstract: The DNA sequence of about 5.9 kilobase pairs (kbp) of the gramicidin S biosynthesis operon (grs) was determined. Three open reading frames were identified; the corresponding genes, called grsT, grsA, and grsB, were found to be organized in one transcriptional unit, not two as previously reported (M. Krause and M. A. Marahiel,  1988). The entire nucleotide sequence of grsA, coding for the 126.663-kilodalton gramicidin S synthetase 1, grsT, encoding a 29.191-kilodalton protein of unknown function, and 732 bp of the 5' end of grsB, encoding the gramicidin S synthetase 2, were determined. A single initiation site of transcription 81 bp upstream of the grsT initiation condon GTG was identified by high-resolution S1 mapping studies. The sequence of the grsA gene product showed a high degree of homology to the tyrocidine synthetase 1 (TycA protein), and that of grsT exhibited a significant degree of homology to vertebrate fatty acid thioesterases.

title: The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains
abstract: The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.

title: Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping
abstract: Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure.

title: Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis.
abstract: Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial operons encoding peptide synthetases is also discussed.

title: Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA (*)
abstract: Analysis of the primary structure of peptide synthetases involved in non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure. These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation. To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His6-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATPdependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA-His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His). However, it retained the functions of specific amino acid activation and thioester formation. The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP-dependent activation as well as in thioester formation and racemization.

title: The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates*
abstract: Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4′-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a “multiple carrier model” of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

title: The Modular Organization of Multifunctional Peptide Synthetases
abstract: Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme–inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4′-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the “Multiple Carrier Model” of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

title: Multienzymatic non ribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation.
abstract: Peptide synthetases are multienzymatic complexes that synthesize bioactive peptides molecules by the thiotemplate mechanism. Comparison of the known sequences of peptide synthetases led us to the identification of a 350 amino acids domain catalysing elongation and containing the motif HHxxxDG. This motif is present as many times as acyltransfer or epimerisation reactions occur during biosynthesis of the peptide. The distance between this motif and the phosphopantetheinyl attachment site is nearly invariant. An identical motif is found in other enzymes effecting acyl transfer such as chloramphenicol acetyltransferase from Tn9 and dihydrolipoamide acyltransferase. Altogether, the HHxxxDG motif may constitute the signature of a superfamily sharing a common catalytic mechanism based on the acid-base properties of the second histidine for effecting acyl transfer or peptide epimerisation.

title: Peptide Bond Formation in Nonribosomal Peptide Biosynthesis
abstract: Recently, considerable insight has been gained into the modular organization and catalytic properties of nonribosomal peptide synthetases. However, molecular and biochemical aspects of the condensation of two aminoacyl substrates or a peptidyl and an aminoacyl substrate, leading to the formation of a peptide bond, have remained essentially impenetrable. To investigate this crucial part of nonribosomal peptide synthesis, an in vitro assay for a dipeptide formation was developed. Two recombinant holomodules, GrsA (PheATE), providing d-Phe, and a C-terminally truncated TycB, corresponding to the first, l-Pro-incorporating module (ProCAT), were investigated. Upon combination of the two aminoacylated modules, a fast reaction is observed, due to the formation of the linear dipeptided-Phe-l-Pro-S-enzyme on ProCAT, followed by a noncatalyzed release of the dipeptide from the enzyme. The liberated product was identified by TLC, high pressure liquid chromatography-mass spectrometry, 1H and 13C NMR, and comparison with a chemically synthesized standard to be the expectedd-Phe-l-Pro diketopiperazine. Further minimization of the two modules was not possible without a loss of transfer activity. Likewise, a mutation in a proposed active-site motif (HHXXXDG) of the condensation domain giving ProCAT(H147V), abolished the condensation reaction. These results strongly suggest the condensation domain to be involved in the catalysis of nonribosomal peptide bond formation with the histidine 147 playing a catalytic role.

title: Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis
abstract: The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo‐bases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

title: Molecular Cloning and Characterization of Fengycin Synthetase Gene fenB from Bacillus subtilis
abstract: A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed inEscherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, aKm value of 922 μM, and a turnover number of 236 s−1. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.

title: Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis
abstract: Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.

title: A putative new peptide synthase operon in Bacillus subtilis: partial characterization.
abstract: A large operon-type structure has been located between the gltA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.

title: Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168.
abstract: A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.

title: Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3.
abstract: Bacillus subtilis strains produce a broad spectrum of lipopeptides that are potent biosurfactants and have specific antimicrobial and antiviral activities. The cyclic lipodecapeptide fengycin is one such compound. Although the fengycin biosynthetic genes in B. subtilis 168 (pps genes) and F29-3 (fen genes) have been well characterized, only limited information is available about the biochemical features of the fengycin synthetase multienzyme system.



Five multifunctional peptide synthetases (Fen1-5) that catalyze biosynthesis of the peptide portion of fengycin have been purified from crude extracts of the B. subtilis b213 and A1/3 strains. These enzymes activate all fengycin amino-acid components as aminoacyl adenylates or aminoacyl thioesters. Fen1, Fen2 and Fen3 are each approximately 286 kDa, Fen4 is approximately 400 kDa and Fen 5 is approximately 140kDa; each enzyme activates a different set of L-amino acids. A five-gene cluster (fen1-5) was detected in the B. subtilis A1/3 genome that shows high homology to the pps and fen genes in B. subtilis strains 168 and F29-3. Disruption of fen4 resulted in a loss of fengycin production. The fengycin synthetase enzymes isolated from B. subtilis b213 were assigned to the corresponding A1/3 fen genes by their amino-terminal sequences.



The structural and functional organization of the fengycin synthetase system from B. subtilis b213 has been characterized in detail and correlated with the corresponding pps and fen genes in B. subtilis strains 168, A1/3 and F29-3. Biosynthesis of the peptide part of fengycin involves five multifunctional modular proteins that assemble the lipopeptide chain using a nonribosomal, multiple carrier thiotemplate mechanism.

title: A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo.
abstract: Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.

title: Construction and Properties of Escherichia coli Strains Exhibiting α-Complementation of β-Galactosidase Fragments In Vivo
abstract: In vivo α-complementation of β-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F′M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F′M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable β-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of β-galactosidase activity.

title: Construction of Tn917ac1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes.
abstract: A Tn917 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes. This transposon, Tn917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn917, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the erm-tnpR-tnpA gene cluster of Tn917 and a temperature-sensitive ori of pE194. For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin. Cells with a Tn917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917. DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli. Sequence analysis revealed that Tn917ac1 was integrated into the chromosome of B. subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain. Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.

title: Experiments in molecular genetics
abstract: Molecular Genetics (Biology): An Overview | Sciencing Experiments in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ... Experimental Molecular Genetics | Biology | MIT OpenCourseWare DNA experiments you can perform at home | SBS Science Experiments in molecular genetics Jeffrey H. Miller ... DNA and Molecular Genetics Experiments in Molecular Biology: Biochemical Applications ... Molecular Genetics Biology Experiment Please help ... Molecular genetics | biology | Britannica Molecular Genetic Experiment : Biology Lab 1793 Words ... Miller, J.H. (1972) Experiments in Molecular Genetics ... Griffith's experiment Wikipedia DNA as genetic material: Revisiting classic experiments ... Experiments in molecular genetics (Book, 1972) [WorldCat.org] Measuring βGalactosidase Activity in Bacteria: Cell ... Classic Experiments in

title: Molecular Cloning: A Laboratory Manual
abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

title: Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917.
abstract: The Streptococcus faecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAM alpha 1::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SP beta. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAM alpha 1::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.

title: [45] Tyrocidine synthetase system
abstract: Publisher Summary This chapter discusses the purification and assay of tyrocidine-synthesizing enzymes. Growing procedure for obtaining enzyme-rich bacillus brevis is presented in the chapter. It is suggested that for a good yield of active enzymes, it is crucial to harvest the organism at the time of maximum enzyme production, and keen attention should be paid to the detection of this time. Purification of tyrocidine-synthesizing enzymes involves preparation of extracts, sephadex G-200 filtration, purification of the light enzyme, purification of the intermediate enzyme, and purification of the heavy enzyme. Of the three complementary enzymes of tyrocidine synthesis, the intermediate and heavy enzymes are polyenzymes, each composed of amino-acid activating subunits of 70,000 molecular weight and of a 4'-phosphopantetheine-containing protein of 17,000 molecular weight, which carries the growing nascent peptides. The dissociation of these polyenzymes may be achieved either by extended incubation of crude bacterial extracts or by incubating the purified polyenzymes with a Triton extract made from the insoluble fraction of the bacterial lysate.

title: Substrate Specificity of Hybrid Modules from Peptide Synthetases*
abstract: Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences. The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein. The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed. We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG. The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity. Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units. The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate. For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues. The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules.

title: Nucleotide sequences that signal the initiation of transcription and translation inBacillus subtilis
abstract: SummaryWe have determined the nucleotide sequence of twoBacillus subtilis promoters (veg andtms) that are utilized by the principal form ofB. subtilis RNA polymerase found in vegetative cells (σ55-RNA polymerase) and have compared our sequences to those of several previously reportedBacillus promoters. Hexanucleotide sequences centered approximately 35 (the “-35” region) and 10 (the “-10” region) base pairs upstream from theveg andtms transcription startpoints (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed toEscherichia coli promoters. Conformity to the preferred -35 and -10 sequences may not be sufficient to promote efficient utilization byB. subtilis RNA polymerase, however, since three promoters (veg, tms andE. coli tac) that conform to these sequences and that are utilized efficiently byE. coli RNA polymerase were used with highly varied efficiencies byB. subtilis RNA polymerase.We have also analyzed mRNA sequences in DNA located downstream from eightB. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3′ terminal region ofB. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.