bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae
Authors: Potts W. J. , Saunders J. R.
Published: October 2006

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that areantigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7amino acid N‐terminal pre‐pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N‐methylphenylalanine pili in general. The succeeding N‐terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino‐terminus of atleast one type of meningococcal pilin. Other regions that are conserved in variant pilin potypeptides from Neisseria gonorrhoeae are conserved at the aminoacid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pH sequence. Sequences extending downstream for about 140bp on the 3′ side of the coding region for both pilin genes are only about 85% homologous.
Epitope mapping with solid‐phase peptides: identification of type‐, subspecies‐, species‐ and genus‐reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis
Authors: Conlan J. W. , Clarke I. N. , Ward M. E.
Published: October 2006

The major outer membrane protein (MOMP) of Chlamydiatrachomatis carries serovar‐, subspecies‐, species‐ and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different servoars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero‐taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovarL1 MOMP
High‐level expression and epitope localization of the major outer membrane protein of Chlamydia trachomatis serovar L1
Authors: Pickett M. A. , Ward M. E. , Clarke I. N.
Published: October 2006

Fragments of the gene encoding the major outer membrane porin protein (MOMP) of Chlamydia trachomatis serovar L1 were ligated into the pUC plasmid vectors to give a series of overlapping recombinant sex pressing MOMP from the lac promoter. Induction of this promoter with IPTG leads to high‐level expression of the recombinant porin protein. Electron microscopy shows the presence of insoluble inclusions within the Escherichia coli host cells. Probing the expressed MOMP fragments with a set of monoclonal antibodies permitted localization of the four binding sites (epitopes)of primary‐sequence‐dependent monoclonal antibodies that exhibit genus‐, species‐, subspecies and type (serovar)‐specific reactivities.
Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis
Authors: Perego M. , Spiegelman G. B. , Hoch J. A.
Published: October 2006

Sporulation begins coincidentally with the expression of several stationary‐phase‐associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary‐phase‐associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96‐amino‐acid protein (molecular weight 10773) and contained a helix‐turn‐helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB 4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB 15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.
Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis
Authors: Honeyman A. L. , Stewart G. C.
Published: October 2006

The rodC 1 mutation of Bacillus subtilis is a temperature‐sensitive marker which affects the orientation of the plane of cell division. We have cloned the rodC gene and have localized the site of the rodC 1 lesion. To identify the rodC gene product, we have subjected several plasmid clones containing B. subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli. A 68 kiloDalton protein has been identified as the rodC gene product. This is the initial cloning of a cell division gene and the identification of its product from B. subtilis. The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid.
Nucleotide sequence of the ugp genes of Escherichia coli K‐12: homology to the maltose system
Authors: Overduin P. , Boos W. , Tommassen J.
Published: October 2006

The nucleotide sequence of the ugp genes of Escherichia Coli K‐12, which encode a phosphate‐limitation inducible uptake system for sn‐glycerol‐3‐phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1‐nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein‐dependent transport systems, but they seem to be particularly closely related to the maltose system.
Three copies of a single protein II‐encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication
Authors: van der Ley P.
Published: October 2006

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II‐encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc‐encoding gene might have been generated by both gene conversion and gene duplication.
Three α‐amylase genes of Aspergillus oryzae exhibit identical intron‐exon organization
Authors: Wirsel S. , Lachmund A. , Wildhardt G. , Ruttkowski E.
Published: October 2006

We have cloned three genes (amy1, amy2 and amy3) encoding α‐amylase in the filamentous fungus Aspergillus oryzae. The established overall sequences have a very high degree of homology, showing divergences mainly in the 3’‐untranslated regions. The positions and the sequences of the eight introns were found to be absolutely identical in the three genes. The sequence analysis of the 5’‐regions revealed presumptive TATA, CAAT and GC boxes. Primer extension analysis was performed to determine the transcription start. We were able to detect mRNAs from amy1 and amy3 but not from amy2 with gene‐specific oligonucleotide probes complementary to the 3’‐noncoding regions.
Transformation of Mycobacterium smegmatis with Escherichia coii plasmids carrying a selectable resistance marker
Authors: Zainuddin Z. F. , Kunze Z. M. , Dale J. W.
Published: October 2006

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol‐resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re‐transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.
Partial nucleotide sequence of the pts operon in Salmonella typhimurium: comparative analyses in five bacterial genera
Authors: Schnierow B. J. , Yamada M. , Saier M. H.
Published: October 2006

The nucleotide sequence of a Salmonella typhimurium DNA segment of 549 base pairs which encompasses the operator‐promoter of the pts operon, the entirety of the ptsH gene, encoding HPr of the phosphotransferase system (PTS), the first 29 nucleotides of the ptsl gene, encoding Enzyme I of the PTS, and the intercistronic region between the ptsH and ptsl genes was determined and compared with the corresponding sequence from Escherichia coli (De Reuse et al., 1985). The two sequences showed 91 % overall identity, with some regions showing sequence conservation and others exhibiting relative divergence. Two open reading frames were identified in both species: one encoded HPr on the ‘sense’ strand (255 nucleotides; 12 nucleotide differences, no amino acid differences); the other, on the anti‐sense strand, consisted of 291 nucleotides (13 nucleotide differences, 13 amino acid differences). While HPr bears a net negative charge, the putative protein encoded by the open reading frame on the anti‐sense strand is strongly basic.

Computer analyses of HPr proteins from five different bacterial genera revealed four regions which show strong sequence identity and therefore are presumed to be critical for maintenance of biological activity. Two of these regions were specific to Gram‐positive bacteria. Proposed functions for each of these regions are discussed. Relative evolutionary distances between the HPr proteins were also computed.
Genetics of pertussis toxin
Authors: Gross R. , Aricò B. , Rappuoli R.
Published: October 2006

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.
Identification of amino acid sequences that can function as translocators of β‐lactamase in Escherichia coli
Authors: Zhang Y. , Broome‐Smith J. K.
Published: October 2006

A plasmid vector, pYZ1, was constructed which lacks most of the β‐lactamase signal‐peptide coding region, but has a unique EcoRI site spanning codons 2 and 3 of the resultant cytoplasmic β‐lactamase derivative. Short quasi‐random DNA sequences were cloned into the EcoRI site and Escherichia coli transformants in which some translocation of β‐lactamase across the cytoplasmic membrane was restored were selected by their ability to survive and form colonies on plates containing a low level of ampicillin. About 15–20% of all in‐frame inserts restored some β‐lactamase trans‐location and the salient feature of these sequences was their marked hydrophobicity. These results are discussed in the light of a similar study in which sequences able to function as translocators of invertase in yeast were cloned and analysed (Kaiser et al., 1987).
Subcellular localization of proteins encoded by the phenotypically cryptic plasmid of Neisseria gonorrhoeae: biological evidence for outer membrane association of the cppB gene product
Authors: Aalen R. B. , Lossius I. , Gundersen W. B.
Published: October 2006

Almost all clinical isolates of Neisseria gonorrhoeae harbour a plasmid of 4.2 kb with no known function. A genetic model based on the DNA sequence of the plasmid, with ten open reading frames, has been proposed by Korch et al., (1985). To address the question of the function of the encoded proteins, some of which are expressed when the plasmid is harboured by Escherichia coli, the subcellular locations of such proteins were investigated in minicells of Escherichia coli DS410. The protein CppB, earlier proposed to be a membrane‐spanning polypeptide, was found associated with the outer membrane. Up to five other cryptic plasmid proteins were found to be localized in the periplasm.
The gene for a small stable RNA (10Sa RNA) of Escherichia coli
Authors: Chauhan A. K. , Apirion D.
Published: October 2006

A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3′ end. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.
Isolation of α‐tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of α‐tubulin
Authors: Holloway S. P. , Sims P. F. G. , Delves C. J. , Scaife J. G. , Hyde J. E.
Published: October 2006

As a step towards indentifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire α‐tubulin gene (designated α‐tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other α‐tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the α‐tubulin I gene itself was found, although a second gene designated α‐II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The α‐I and β‐tubulin genes were found to reside on different chromosomes.
The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE
Authors: Downie J. A.
Published: October 2006

The predicted protein sequence of the nodL gene from Rhizobium leguminosarum was screened against translations of the GenBank DNA sequence database. A very strong homology was found to lacA, which encodes thiogalactoside transferase; homology between NodL and the cysE gene product (serine acetyl transferase) was also found. Comparison of the conserved regions of the three protein sequences indicated a domain that may be an active site of the enzymes.
Families of bacterial signal‐transducing proteins
Authors: Gross R. , Aricò B. , Rappuoli R.
Published: October 2006

Bacteria can respond to a variety of environmental stimuli by means of systems generally composed of two proteins. The first protein (sensor or transmitter) is usually a transmembrane protein with cytoplasmic and extracytoplasmic domains. The extracytoplasmic domain (sensor) senses the environment and transfers the signal through the transmembrane domain to the cytoplasmic domain (transmitter), which has kinase activity. The second protein is located in the cytoplasm and contains an amino‐terminal domain (receiver), which can be phosphorylated by the transmitter, and a carboxy‐terminal region (regulator), which regulates gene expression by binding to DNA. The transmitter and receiver modules (the kinase and its target) are conserved in all signal‐transducing systems and are the‘core structure’of this two‐component system. The sensors and the regulators vary according to the stimuli they respond to and the DNA structure they interact with. On the basis of their sequence homology, the proteins belonging to such two‐component systems can be classified into different families, which are summarized in this review.
Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus
Authors: Monack D. M. , Arico B. , Rappuoli R. , Falkow S.
Published: October 2006

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the iocus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir−) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir− phase variants. The vir locus and the adjacent 5′ portion of the fhaB structural gene were cloned from the parental Vir+B. bronchiseptica strain on a 23.5kb BamHl fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir− variants of Bordetella pertussis and B. bronchiseptica as well as wr::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.
Functional characterization of a putative internal promoter sequence between the 16S and the 23S RNA genes within the Escherichia coli rrnB operon
Authors: Zacharias M. , Wagner R.
Published: October 2006

Transcription of ribosomal RNAs in Escherichia coli is started from two strong tandem promoters, P1 and P2. tt is known, however, that internal promoter‐like structures occur and in a recent report (Mankin et al., 1987) a promoter sequence Pi within the 16S and 23S RNA spacer region showing good homology to the pro‐karyotic consensus promoter structure was identified. It was proposed that this putative promoter has a possible function in the transcription of ribosomal RNAs in E. coli. Fusion of various DNA fragments containing the putative promoter sequence and different parts of the 16S/23S spacer region as well as the 23S RNA to the galactokinase gene allowed us to assess the functional activity of the promoter in vivo. To determine any growth rate dependent function of the putative promoter, the measurements were performed under different growth conditions. The promoter activity did not exceed 7% of the lac promoter under in vivo assay conditions. In addition, transcription starting at the promoter Pi did not proceed through the entire 23S RNA gene. We conclude, therefore, that transcription from Pi does not contribute significantly to the synthesis of ribosomal RNAs. Thus Its functional significance, if any, remains elusive.
The biochemical basis for growth inhibition by L‐phenylalanine in Neisseria gonorrhoeae
Authors: Bhatnagar R. K. , Berry A. , Hendry A. T. , Jensen R. A.
Published: October 2006

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L‐phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L‐tyrosine or 4‐hydroxyphenylpyruvate. Phenylalanine‐resistant (PheR) and phenylalanine‐sensitive (Phes) isolates both have a single 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase that is partially inhibited by L‐phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4‐hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects, (i) Phenylalanine decreases precursor flow to 4‐hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4‐hydroxyphenylpyruvate to L‐tyrosine.
Integration of the Serratia marcescens haemolysin into human erythrocyte membranes
Authors: Schiebel E. , Braun V.
Published: October 2006

The haemolytic activity of Serratia marcescens is determined by two proteins, ShIA and ShIB. ShIA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShIA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShIA by added trypsin. Our results suggest that the conformation of inactive ShIA (from an ShIB− strain) differs from the active ShIA, and that in a hydrophobic environment (detergent or membrane) active ShIA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShIA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShIA integrated into ‘ghosts’ remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C‐terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte ‘ghosts’, we conclude that ShiA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water‐filled channel and is resistant to trypsin and other proteases.
The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth
Authors: Bétermier M. , Lefrère V. , Koch C. , Alazard R. , Chandler M.
Published: October 2006

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site‐specific recombinational switches. Furthermore, we show that induction of a Mucte62 prophage in a fis lysogen occurs at a lower temperature than that of a wild‐type strain, and that spontaneous induction of Mucfs62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage λ.
Sequence analysis of the wall‐associated protein precursor of Streptococcus mutans antigen A
Authors: Ferretti J. J. , Russell R. R. B. , Dao M. L.
Published: October 2006

The nucleotide sequence has been determined for the Streptococcus mutans wall‐associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45276) for strain Ingbritt and 416 amino acids (Mr, 44846) for strain GS5. In the C‐terminal region of the wall‐associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram‐positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29000 into the culture supernatant. Thus, the wall‐associated protein A is a precursor of the 29000 Mr antigen A.
Klebsiella pneumoniae strain K21: evidence for the rapid secretion of an unacylated form of pullulanase
Authors: Kornacker M. G. , Boyd A. , Pugsley A. P. , Plastow G. S.
Published: October 2006

Klebsiella pneumoniae strain PAP996 was previously shown to secrete fatty acylated, aggregated (micellar) pullulanase only after the end of exponential growth. Here we show that the closely related strain K21 secretes large amounts of unacylated, non‐aggregated (monomeric) pullulanase during exponential growth. Only a small amount (<10%) of the secreted pullulanase was initially retained by the exponentially growing cells to be subsequently secreted in a fatty acylated, aggregated form. Despite the absence of fatty acids in secreted monomeric pullulanase, the effects of the antibiotic globomycin on pullulanase maturation indicated that all of the enzyme synthesized by strain K21 is processed by lipoprotein signal peptidase.
Characterization of the neisserial lipid‐modified azurin bearing the H.8 epitope
Authors: Woods J. P. , Dempsey J. F. , Kawula T. H. , Barritt D. S. , Cannon J. G.
Published: October 2006

The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 Mab‐binding protein with a consensus lipoprotein processing site, an N‐terminal domain containing the epitope for H.8 MAb binding, and a C‐terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin‐sensitive processing, and that it was immunologically cross‐reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti‐azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.
Construction of lac fusions to the inducible arginine‐and lysine decarboxylase genes of Escherichia coli K12
Authors: Auger E. A. , Redding K. E. , Plumb T. , Childs L. C. , Meng S.‐Y. , Bennett G. N.
Published: October 2006

The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of β‐galactosidase expression. The position of the Mud‐lac fusion in lysine decarboxylase‐deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.
Molecular characterization and sequence of phosphatidylinositol‐specific phospholipase C of Bacillus thuringiensis
Authors: Lechner M. , Kupke T. , Stefanovic S. , Götz F.
Published: October 2006

The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI‐specific phospholipase C, PI‐PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38095). The NH2‐terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI‐PLC consists of 299 amino acid residues with a molecular weight of 34586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA‐donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingo‐myelin. The cleaving specifity of PI‐PLC was examined by thin layer chromatography.
Topographical alterations in proteins I of Neisseria gonorrhoeae correlated with lipooligosaccharide variation
Authors: Judd R. C. , Shafer W. M.
Published: October 2006

Four transformant strains of Neisseria gonorrhoeae were generated, two of which (WS3 and WS5) had protein I subclass A (P.IA) and two which (WS2 and WS4) had protein I subclass B (P.IB). Analysis of the strains demonstrated that the two P.IA‐bearing strains differed in lipooligosaccharide (LOS) and H.8 antigen, as assessed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immuno‐blotting. The WS5 strain had slow‐migrating LOS and H.8 antigen, and the WS3 strain had fast‐migrating LOS and H.8 antigen. The P.IB‐bearing strains also had either slow‐migrating LOS and H.8 antigen (WS4) or fast‐migrating LOS and H.8 antigen (WS2). Structural and exposure analysis revealed that although the P.IAs were identical in the WS3 and WS5 strains, there was a slight alteration of the exposure of the proteins which correlated with altered LOS and/or H.8 antigen. The P.IBs were also shown to be structurally identical, but the LOS and/or H.8 antigen variation in these strains correlated with a more pronounced alteration in the exposure of the P.IB molecules. The differences in protein I (P.I) exposure were generally found in highly negatively charged regions of the molecule, suggesting that the immunogenicity and/or antigenicity of the P.I molecules may vary as a result of LOS and/or H.8 antigen alterations.
The proline transport protein of Aspergillus nidulans is very similar to amino acid transporters of Saccharomyces cerevisiae
Authors: Sophianopoulou V. , Scazzocchio C.
Published: October 2006

In Aspergillus nidulans, the gene prnB encoding the major proline transport system is one of a cluster of four genes necessary and sufficient for the utilization of proline as sole nitrogen and/or carbon source. The prn cluster has been cloned and the sequence and transcript map of the prnB gene are presented in this paper. The predicted translated sequence consists of 570 amino acids, resulting in a molecular weight of 63028 Daltons. Its hydropathy profile shows 10 hydrophobic segments typical of integral membrane proteins. No N‐terminal hydrophobic signal peptide is present, the N‐terminal and C‐terminal ends of the protein being hydrophilic. Similar results were previously found for the arginine and histidine transporters of Saccharomyces cerevisiae, with which the prnB transporter shares regions of highly conserved amino acid sequences. Using S1 mapping and Northern blot analyses, we confirm the presence of a unique inducible prnB transcript of 1.9 kb.
A Yersinia pestis‐specific DNA fragment encodes temperature‐dependent coagulase and fibrinolysin associated phenotypes
Authors: McDonough K. A. , Falkow S.
Published: October 2006

The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature‐dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis‐specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature‐dependent: coagulase activity is most evident at temperatures below 30°C but fibrinolytic activity increases with higher temperatures (>30°C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post‐translationally. It is possible that the alternative forms of the Pla protein are essential to ‘flea blockage’ and subsequent transmission of the plague bacillus to animals.
Immunity protein is not required for the bactericidal activity of colicin E3
Authors: Escuyer V. , Perrin D. , Mock M.
Published: October 2006

Plasmid pLAX3, carrying the colicin E3 gene, was used to direct the in vitro synthesis of a colicin E3*molecule totally devoid of its immunity protein. We established that this molecule is able to kill sensitive Escherichia coli cells in the total absence of immunity protein. Therefore, all of the information required for colicin E3 action is located on the colicin potypeptide itself. Furthermore, our studies indicated that immunity protein protects the C‐terminal enzymatic part of native colicin E3 protein against proteolytic degradation before or during its translocation across the cell envelope. These results are discussed in relation to the mode of entry of colicin E3 into bacterial cells.
Analysis of C4‐dicarboxylate transport genes in Rhizobium meliloti
Authors: Yarosh O. K. , Charles T. C. , Finan T. M.
Published: October 2006

A 5.1 kbp DNA fragment was isolated which complemented C4‐dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA‐generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second‐site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen‐fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.
Detection and identification of mycobacteria by amplification of mycobacterial DNA
Authors: Hance A. J. , Grandchamp B. , Lévy‐Frébault V. , Lecossier D. , Rauzier J. , Bocart D. , Gicquel B.
Published: October 2006

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Tag polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 106 human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.
Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid‐borne lacZ fusions
Authors: Gerstel B. , McCarthy J. E. G.
Published: October 2006

The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and β‐galactosidase activities encoded by the atp:: lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans‐acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible CiS and trans regulatory phenomena.
Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of activation of the protoxin
Authors: Hayashi T. , Kamio Y. , Hishinuma F. , Usami Y. , Titani K. , Terawaki Y.
Published: October 2006

The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non‐producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host‐range vector plasmid.
Genetic factors influencing lignin peroxidase activity in Phanerochaete chrysosporium ME446
Authors: Raeder U. , Thompson W. , Broda P.
Published: October 2006

Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP‐mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase‐active state within the idiophasic mycelial mat and may be the mating‐type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.
A Mycoplasma genetic element resembling prokaryotic insertion sequences
Authors: Ferrell R. V. , Heidari M. B. , Wise K. S. , McIntosh M. A.
Published: October 2006

Nucleotide sequence analysis of two Mycoplasma hyopneumoniae‐derived copies of a repetitive genetic element revealed structural similarities to typical prokaryotic insertion sequences. This is the first such sequence identified in the class Mollicutes. The element spans approximately 1550bp, with 28bp inverted terminal repeats. Two open reading frames occur within the sequence, one potentially encoding a protein with a size‐variant alpha‐helical domain containing heptameric leucine periodicity. Hybridization data with several strains from each of two mycoplasma species showed that the repetitive sequence is variably distributed within the M. hyopneumoniae and Mycoplasma hyorhinis chromosomes and indicated that in some cases the repeated sequence is contained within a larger genetic element which may be the result of phage or plasmid Insertion.
Analysis of the chromosomal location of two copies of a Bordetella pertussis insertion sequence
Authors: McPheat W. L. , Hanson J. H. , Livey I. , Robertson J. S.
Published: October 2006

IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481V1 is located within 3kbp of the start of the adenylate cyclase gene whilst IS481v2 is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481 v1 or IS481v2.
Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems
Authors: Stirling D. A. , Hulton C. S. J. , Waddell L. , Park S. F. , Stewart G. S. A. B. , Booth I. R. , Higgins C. F.
Published: October 2006

The proU loci of Salmonella typhimurium and Escherichia coli encode high‐affinity glycine betaine transport systems which play an important role in survival under osmotic stress. Transcription of the proU locus is tightly regulated by osmolarity and this regulation appears to be mediated by osmotically induced changes in DNA supercoiling. In order to study the regulatory mechanisms involved we have cloned and characterized the proU locus of S. typhimurium by an in vivo transductional procedure. The locus is shown to consist of at least three genes, designated proVWX, cotranscribed as a single operon. The first gene in the operon encodes a protein sharing considerable sequence identity with ATP‐binding proteins from other periplasmic transport systems. Unexpectedly, the highly expressed periplasmic glycine betaine binding protein was found to be encoded by a distal gene, proX, in the operon. The operon has no significant internal promoters but is expressed from a single osmoregulated promoter whose transcription start site has been mapped. The proU promoter of E. coli has also been sequenced and the transcription start site shown to be similar to that of S. typhimurium. Evidence is presented which suggests that, besides de novo glycine betaine uptake, an important function of ProU may be the recapture and recycling of other osmolytes that leak from the cell.
Transcriptional polarity enhances the contribution of the internal promoter, ilvEp, in the expression of the ilvGMEDA operon in wild‐type Escherichia coli K12
Authors: Lopes J. M. , Soliman N. , Smith P. K. , Lawther R. P.
Published: October 2006

The ilvG gene of Escherichia coli K12 produces a cryptic peptide as a result of a frameshift mutation located approximately halfway through the coding sequence of the gene. This mutation is polar on expression of the downstream genes (ilvEDA) because transcription terminates within the translationally barren region that results from the mutation. Contrary to this, Salmonella typhimurium produces a full‐length functional ilvG protein and is therefore unlikely to manifest this polarity event. E. coli K12 strains with mutations either in the ilvG gene (which restores a full‐length protein) or in the ρ gene, relieve this polarity suggesting that this event couples transcription and translation in a manner analogous to attenuation. This paper describes experiments designed to determine the molecular nature and location of the polarity event. Most significantly, this work establishes the contribution of the internal promoter (ilvEp, located downstream of the polar site) to the expression of the downstream genes in E. coli K12 wild‐type and mutant strains (ilvG) and by extension to the role of this promoter in S. typhimurium. This analysis suggests that ilvEp contributes as much as 90% of ilvEDA expression in wild‐type E. coli K12 and only 15% In wild‐type S. typhimurium when grown under non‐repressing conditions.
Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens mel operon
Authors: Geistlich M. , Irniger S. , Hütter R.
Published: October 2006

The transcription initiation site of the meloperon from Streptomyces glaucescens, determined by S1 mapping and primer elongation experiments, lies 32 to 34bp upstream of the translation initiation codon of the first open reading frame. A total of 172 to 219bp upstream of the transcription start point are necessary for a fully active and regulated mel promoter. Deletion analysis, gel retardation assays and DNAse I footprint experiments facilitated division of the promoter into three functional domains, which include the RNA polymerase recognition site up to nucleotides ‐33 to ‐42, the binding region of a protein of assumed regulatory function between nucleotides ‐65 and ‐93, and an upstream activator site, located between positions ‐158 and ‐219.
Translational repression of TEM β‐lactamase synthesisas a response of Escherichia coli to heat shock
Authors: Kuriki Y.
Published: October 2006

As a result of a temperature shift‐up from 30°C to 42°C, β‐lactamase synthesis in Escherichia coli carrying pBR322 ceased transiently, even though the level of β‐lactamase mRNA was not altered. pBR328‐directed pre‐β‐lactamase synthesis in an in vitro transcription‐translation coupled system was also repressed by incubation at the higher temperature. Translation of the lacZ sequence from the amp translation start signal, inserted into the open reading frame vector pORF1, was also repressed transiently upon the temperature shift‐up. Pre‐heating of the in vitro coupled system at 45°C specifically reduced its capacity for pre‐β‐lactamase synthesis. This capacity was restored by the addition of a 160000 × g supernatant prepared from E. coli grown at 30°C, but not by the supernatant from the cells incubated at 42°C. These and other results Indicate that (i) the 160000 × g supernatant contains a heat‐labile protein(s) that is required for efficient initiation of the translation of pre‐β‐lactamase mRNA, and (ii) the heat shock‐induced repression of β‐lactamase synthesis is due to inactivation of the protein(s) in the 160000 × g supernatant.