bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of multiple types of N‐methyl Phe pili in Pseudomonas aeruginosa
Authors: Elleman T. C. , Peterson J. E.
Published: October 2006

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid‐borne genes of homologous pilins from Bacteroides nodosus are Introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two seroiogically distinct populations of pili on each cell. Simultaneous production of both Indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of Interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.
Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora
Authors: Hinton J. C. D. , Salmond G. P. C.
Published: October 2006

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram‐negative bacteria. We have used a λ‐sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon‐based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein Is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA‐induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.
Identification and characterization of the transferrin receptor from Neisseria meningitidis
Authors: Schryvers A. B. , Morris L. J.
Published: October 2006

Expression of the meningococcal transferrin receptor, detected by assay with human transferrin conjugated to peroxidase, was regulated by the level of iron in the medium. The transferrin receptor was identified by SDS‐PAGE and Western blot analysis, as a 71 000 molecular weight iron‐regulated outer membrane protein in Neisseria meningitidis B16B6. Growth studies with iron‐deficient cells and competition binding experiments demonstrated that the meningococcal receptor was species‐specific for human transferrin. Reciprocal competitive binding experiments and limited proteolysis of intact cells indicated that the transferrin and lactoferrin receptors are distinct.
Chromosomal mapping of the pel and cel genes in Erwinia chrysanthemi strain B374
Authors: Piecq M. , van Gijsegem F. , Schoonijans E. , Toussaint A.
Published: October 2006

Using the RP4::mini‐Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co‐transfer mapping of Mud11734 insertion mutants and refined the map by co‐transposition analysis. This analysis has enabled us to identify another cel gene.
Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii
Authors: Bennett L. T. , Cannon F. , Dean D. R.
Published: October 2006

The nucleotide sequence of the nifA gene from Azotobacter vinelandii was determined. This gene encodes an Mr= 58100 polypeptide that shares significant sequence identity when compared to nifA‐encoded products from other organisms. Interspecies comparisons of nifA‐encoded products reveal that they all have a consensus ATP binding site and a consensus DNA binding site in highly conserved regions of the respective polypeptides. The nifA gene immediately precedes the nifB‐nifQ gene region but is unlinked to the major nif gene cluster from A. vinelandii. A potential regulatory gene precedes and is apparently cotranscribed with nifA. Mutant strains that have a deletion or a deletion plus an insertion within nifA are incapable of diazotrophic growth and they fail to accumulate nitrogenase structural gene products.
Transcription of a Rhizobium ieguminosarum biovar phaseoli gene needed for melanin synthesis is activated by nifA of Rhizobium and Klebsiella pneumoniae
Authors: Hawkins F. K. L. , Johnston A. W. B.
Published: October 2006

The Rhizobium leguminosarum biovar phaseoli symbiotic plasmid pRP2JI carries a gene, melA, specifying the enzyme tyrosinase, which is responsible for the production of the pigment melanin in these bacteria. Transcription of melA is activated by the nifA gene of Rhizobium and, when the cloned melA gene is transferred to Escherichia coli, melA is expressed if the recipients contain nifA gene of Klebsiella pneumoniae. This nifA‐dependent activation was temperature sensitive and required the ntrA gene. The cloned nifA gene of K. pneumoniae, when transferred to a nifA mutant of Rhizobium phaseoli biovar phaseoli, corrected the Mer− but not the Fix− phenotype. nifA of R. leguminosarum biovar phaseoli activated melA at higher levels in cells grown in low concentrations of oxygen. Also, nifA of fl. leguminosarum biovar phaseoli activated nifH of K. pneumoniae in Escherichia coli cells grown In low‐oxygen concentrations.
Regulation of the virC and virD promoters of pTiC58 by the ros chromosomal mutation of Agrobacterium tumefaciens
Authors: Tait R. C. , Kado C. I.
Published: October 2006

The virC and virD operons of the virulence region of the Ti plasmid are highly regulated, requiring a transcriptional regulator that is encoded by virG and is activated by the product of virA and plant phenolics such as acetosyringone. Full expression of virC and virD of octopine and nopaline Ti plasmids is also obtained by a mutation in the ros gene of the Agrobacterium tumefaciens chromosome. S1‐nuclease analysis, in vitro transcription, and DNase I protection experiments with A. tumefaciens RNA polymerase revealed virD promoters tandemly arranged, both of which are functional in the Ros mutant, while only one is functional in the presence of acetosyringone. A third (overlapping) promoter appears to be responsible for transcription of virC. Expression of virC and virD appears to be modulated by factors within the bacterium by means of a mechanism that is independent of factors produced during infection of the host plant.
Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae
Authors: Schryvers A. B.
Published: October 2006

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in all H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron‐loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron‐loaded lactoferrin and apolactoferrin.
The expression of mutant pilins in Pseudomonas aeruginosa: fifth position glutamate affects pilin methylation
Authors: Pasloske B. L. , Paranchych W.
Published: October 2006

The expression within Pseudomonas aeruginosa PAO1 of three mutant pilin genes from P. aeruginosa PAK was studied to determine their effects on pilin stability, translocation into the membrane, leader peptide removal, and methylation of the mature N‐terminal phenylalanine. The results revealed that a deletion of 4 or 8 amino acids within the immediate N‐terminus of pilin had deleterious effects upon leader peptide cleavage. In addition, while the 4‐amino‐acid deletion did not affect pilin partitioning into the membrane, the 8‐amino‐acid deletion decreased the amount of pilin found within the membrane fraction. Of considerable interest was the finding that the mutation within the mature pitin of the glutamate at position 5 to a lysine did not prevent leader peptide removal but did inhibit the methylation of the N‐terminal phenylalanine.
The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite
Authors: Jayaraman P.‐S. , Gaston K. L. , Cole J. A. , Busby S. J. W.
Published: October 2006

Using recombinant DNA techniques, nested deletions have been made upstream of the Escherichia coli nirB transcription start site and their effects on the regulation of nirB promoter activity have been measured. Nucleotide sequences downstream of −73 are sufficient for FNR‐dependent induction of activity by anaerobic growth conditions. However, nucleotide sequences between −87 and −149 are essential for further induction by nitrite in the growth medium. The nucleotide sequence at the galP1 CRP binding site located from −31 to −52 displays some similarities with the same region at the nirB promoter. When the galP1 sequence from −30 to −54 was replaced by the corresponding nirB sequence, expression from galP1 became inducible by FNR under anaerobic growth conditions.
Cloning of a novel pilin‐like gene from Bordetella pertussis: homology to the fim2 gene
Authors: Pedroni P. , Riboli B. , de Ferra F. , Grandi G. , Toma S. , Aricò B. , Rappuoli R.
Published: October 2006

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.
Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease
Authors: Ahmad M. , Bussey H.
Published: October 2006

We have examined the topology of the yeast arginine permease, a plasma‐membrane protein with multiple membrane‐spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane‐spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmicreticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence ofglycosylation in a systematic series of such fusions gave an internally consistent model for the lumenat orcytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.
Nucleotide sequence analysis of 2″‐aminoglycoside nucleotidyl‐transferase ANT(2″) from Tn4000: its relationship with AAD(3″) and impact on Tn21 evolution
Authors: Schmidt F. R. J. , Nücken E. J. , Henschke R. B.
Published: October 2006

Aminoglycoside 2″‐O‐nucleotidyltransferase (AAD(2″)) mediates bacterial resistance to dibekacin, gentamicin, kanamycin, sisomicin and tobramycin. Its coding sequence, aadB, is part of Tn21‐related transposon, Tn4000. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 177 amino acids with a calculated molecular weight of 21240. The predicted amino acid sequence revealed up to 27% homology to that of three nucleotidyltransferases of type AAD(3″), which are widely distributed among Gram‐negatives, and to the AAD(9) from Staphylococcus aureus transposon Tn554. The regions flanking aadB suggest that its insertion into Tn21 arose from a site‐specific recombination event adjacent to the aadA gene.
Comparison of the chemotactic behaviour of Rhizobium leguminosarum with and without the nodulation plasmid
Authors: Armitage J. P. , Gallagher A. , Johnston A. W. B.
Published: October 2006

The chemotactic behaviour of a strain of Rhizobium leguminosarum biovar viciae was investigated. The flavanoids apigenin and naringenin, inducers of transcription of the nodulation (nod) genes, were both potent attractants but hesperitin, another flavone nod gene inducer, was not. The response of strains containing the Sym plasmid pRL1JI to apigenin and naringenin was significantly greater than the response of a strain cured of the plasmid, although both strains gave a positive response. Addition of the flavanol kaempferol, an antagonist of nod gene induction, had no detectable effect on the chemotactic response to naringenin or aplgenin, but was itself found to be an attractant. The attractant response to a variety of amino acids and sugars was not affected by the presence of the Sym plasmid. Homoserine, the most abundant nitrogenous compound in legume exudates, was also found to be an attractant. However, although the Sym plasmid is required for the biovar to metabolize homoserine as a carbon source, it was not required for the chemotactic response. A group of membrane proteins showed increased methylation in response to stimulation with serine. There was no measurable change in methylation after stimulation with apigenin.
Probing the surface of Neisseria gonorrhoeae: immunoelectron microscopic studies to localize cyanogen bromide fragment 2 in gonococcal pili
Authors: Robinson E. N. , Clemens C. M. , Schoolnik G. K. , McGee Z. A.
Published: October 2006

Common epitopes accessible to antibody on purified macromolecules or structurally altered gonococci may not be accessible to antibody when those macro‐molecules are in their native state on the surface of intact organisms. To determine the immunologic accessibility of cyanogen bromide fragment 2 (CNBr2), a portion of the gonococcal pilin molecule that is common to all gonococcal strains on the surface of viable gonococci, probes composed of specific CNBr2 antibodies linked to gold spheres were manufactured. When whole piliated gonococci were exposed to these anti‐CNBr2 immunological probes and examined using transmission electron microscopy, no significant marking of native pili was evident. These probes, however, detected CNBr2 in purified form. The epitopes encompassed within the CNBr2 portion of pili appear to be inaccessible to anti‐CNBr2 probes within native gonococcal pili.
Construction, transfer and properties of a novel temperature‐sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus
Authors: Luchansky J. B. , Benson A. K. , Atherly A. G.
Published: October 2006

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram‐positive bacteria. The use of a restriction‐deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325‐4. Plasmid pPQ126 (13.6 kb) is a novel, temperature‐sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30°C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39°C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination‐deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30°C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.
Structural and functional analysis of the mini‐circle, a transposable element of Streptomyces coelicolor A3(2)
Authors: Henderson D. J. , Lydiate D. J. , Hopwood D. A.
Published: October 2006

The mini‐circle is a transposable element which is present in Streptomyces coelicolor A3(2) in both free circular and chromosomally integrated linear forms. The nucleotide sequences of the mini‐circle and its preferred site of integration in the Streptomyces lividans TK64 chromosome were determined. Three putative open reading frames were identified in the mini‐circle sequence. The mini‐circle does not appear to cause a target site duplication on transposition and does not have perfect terminal inverted repeats. The observed site‐specificity of the mini‐circle is not mediated by extensive homology between the element and the chromosomal integration site. Transposition of the mini‐circle into the S. lividans chromosome was demonstrated and found to be some two orders of magnitude less efficient than integration of the circular form of the element, suggesting that the circular form of the mini‐circle might be a normal intermediate in the transposition process.
Unique sequences in region VI of the flagellin gene of Salmonella typhi
Authors: Frankel G. , Newton S. M. C. , Schoolnik G. K. , Stocker B. A. D.
Published: October 2006

The HI (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase‐1 antigen in many other Saimonella species. We cloned the H1‐d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1‐d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non‐S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.
An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii
Authors: Yoganathan T. , Lin H. , Buck G. A.
Published: October 2006

Pulsed‐field gel electrophoresis was used to generate a molecular karyotype of chromosomes from the opportunistic AIDS pathogen, Pneumocystis carinii. P. carinii cysts and trophozoites were isolated from immunosuppressed rats, lysed in situ in agarose blocks, and subjected to orthogonal‐field gel electrophoresis (OFAGE) and contour‐clamped homogeneous‐field gel electrophoresis (CHEF). OFAGE and CHEF gels resolved, respectively, 16 or 20 chromosome bands ranging in size from 0.32‐1.5 megabase pairs. Summation of the estimated sizes of these chromosomes suggested a total genome complexity for P. carinii of 8‐16 megabase pairs. Homologous probes for the genes encoding the 18S, 5.8S, and 5S ribosomal RNAs were hybridized to filter blots of the pulsed‐field gels to map these genes to specific P. carinii chromosomes.
A novel family of potentially mobile DNA elements encoding site‐specific gene‐integration functions: integrons
Authors: Stokes H. W. , Hall R. M.
Published: October 2006

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3′ to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3′ and 5′ conserved regions of the DNA element have been determined. A 59‐base sequence element, found at the junctions of inserted DNA sequences and the conserved 3′ segment, is also present at this location in the R46 sequence. A copy of one half of this 59‐base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site‐specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5′ conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site‐specific integration functions (the Integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.
Functional analyses of Proteus mirabilis wild‐type and mutant RecBCD enzymes in Escherichia coli reveal a new mutant phenotype
Authors: Weichenhan D. , Wackernagel W.
Published: October 2006

Cloned into Escherichia coli the recB, recC and recD genes of Proteus mirabilis produce a recBCD enzyme (exoV) functional in recombination and DNA repair. The direction of transcription of recB, recC and recD, the sizes of the enzyme subunits, and their composition in the active enzyme are similar to that observed for the E. coli enzyme. In lambda crosses, the P. mirabilis enzyme has only about 40% of the Chi activity of the E. coli enzyme. The recBCD genes were also cloned from an exoV mutant of P. mirabilis which is u.v.‐sensitive and partly deficient in exoV. The defect was attributed to the recB gene by complementation studies. In a recBCD deletion strain of E. coli, the enzyme from the mutant produced 40% of conjugational recombinants and had retained about 25% of Chi activity. However, it did not restore normal DNA repair, cell viability or recombination in lambda crosses and P1 transduction. The new mutant phenotype is discussed in the light of the assumption that prokaryotic recBCD enzymes can promote recombination in a Chi‐dependent and a Chi‐independent manner.
A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli
Authors: Broome‐Smith J. K. , Bowler L. D. , Spratt B. G.
Published: October 2006

The feasibility of using a β‐lactamase fusion approach for maximizing the levels of periplasmic or membrane‐bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM β‐lactamase was fused after the signal peptide and amino‐terminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3*gene of the unmutagenized I β‐lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a β‐lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.
Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi
Authors: Ji J. , Hugouvieux‐Cotte‐Pattat N. , Robert‐Baudouy J.
Published: October 2006

The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant‐tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non‐secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade‐22 marker on the E. chrysanthemi 3937 chromosome. An R‐prime containing a chromosomal DNA insert of about 30 kb was first obtained; subcloning into pBR325 permitted the isolation of a 4 kb Cial/Sspl fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5 kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the Cial/Sspl fragment demonstrated that oufJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.
Molecular analysis of linear plasmid‐encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi
Authors: Bergström S. , Bundoc V. G. , Barbour A. G.
Published: October 2006

The ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi. The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29334, and (OspB) 296 amino acids long with a molecular weight of 31739. The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event. Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins. These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines.
Expanded linkage map of Erwinia chrysanthemi strain 3937
Authors: Hugouvieux‐Cotte‐Pattat N. , Reverchon S. , Robert‐Baudouy J.
Published: October 2006

In this paper we describe the chromosomal location of various loci in Erwinia chrysanthemi strain 3937. Auxotrophic markers were obtained by chemical mutagenesis, antibiotic resistances were isolated spontaneously and mutations in sugar utilization were obtained by means of Mu insertions. These markers were located on the genetic linkage map of strain 3937 by using a conjugative system mediated by RP4::mini‐Mu plasmids which permitted transfer of genetic material from any point of origin. The location of these markers was compared to that of previously located mutations. Many genes involved in pectinolysis were also located on the E. chrysanthemi 3937 map. These results permitted us to present a new genetic map containing 61 markers distributed over 34 widely scattered loci on the chromosome. Some pairs of markers giving high cotransfer frequencies were tested for cotransduction mediated by the generalized transducing phage phi‐EC2; nine cotransducing pairs were found. It appears that the chromosomal locations of many of these loci are quite different to those of the well‐known enterobacterium Escherichia coli but seem similar to those described for other E. chrysanthemi strains.
Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries
Authors: Okahashi N. , Sasakawa C. , Yoshikawa M. , Hamada S. , Koga T.
Published: October 2006

The complete nucleotide sequence of the gene for a cell‐surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine‐Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N‐terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.
Topology and function of the integral membrane protein conferring immunity to colicin A
Authors: Geli V. , Baty D. , Pattus F. , Lazdunski C.
Published: October 2006

The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore‐forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N‐and C‐terminal regions were found to be directed towards the cytoplasm. Oligonucleotide‐directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.
Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes
Authors: Birkmann A. , Hennecke H. , Bock A.
Published: October 2006

Hybrid 5′ regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF′‐′lacZ or nif′‐′lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF 5’flanking region contains a site recognized by a putative oxygen‐ or nitrate‐responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the nfrA‐dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed In front of the nifD spacer plus promoter from B. Japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH′‐′lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.
The Azorhizobium caulinodans nitrogen‐fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status
Authors: Ratet P. , Pawlowski K. , Schell J. , de Bruijn F. J.
Published: October 2006

The nucleotrde sequence of the Azorhizobium caulinodans ORS571 nlfA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen‐fixing species. Highly conserved domains, including helix‐turn‐helix and ATP‐binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5’upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifAlntr‐mediated control and a consensus element found in the 5’upstream region of the Bradyrhizobium japonicum 5‐aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA‐lac fusion was constructed using miniMu‐lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta.) NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA‐lac expression.
Functional domains of bacteriophage Mu transposase: properties of C‐terminal deletions
Authors: Bétermier M. , Alazard R. , Lefrère V. , Chandler M.
Published: October 2006

We have generated a series of 3’deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the λ Pl promoter, were analysed in vivo for their capacity to complement a superinfecting Mu4am phage, both for lytic growth and lysogeny, and for their effect on growth of wild‐type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.
Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens
Authors: Morel P. , Powell B. S. , Rogowsky P. M. , Kado C. I.
Published: October 2006

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline‐type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine‐type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane‐bound sensor of plant signal molecules.
The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins
Authors: Barlow A. K. , Heckels J. E. , Clarke I. N.
Published: October 2006

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.
Phage‐conversion of cytotoxin production in Pseudomonas aeruginosa
Authors: Hayashi T. , Baba T. , Matsumoto H. , Terawaki Y.
Published: October 2006

We isolated a temperate phage which carried the cytotoxin gene (ctx) from a cytotoxin (CTX)‐producing Pseudomonas aeruginosa strain, PA158. The phage, φCTX, had a head with a hexagonal outline and a contractile tail with tail fibres. The phage genome was a linear double‐stranded 35.5kb DNA with single‐stranded cohesive ends (cos). The attP, cos and ctx genes were all located very close to one another within a 2.3kb segment on the phage genome in the order given (in the circular form). φCTX converted CTX non‐producing P. aeruginosa strains into CTX producers. A single copy of φCTX DNA was integrated at the same site on the host chromosome (attB) in every lysogen, including PA15B. However, the amount of CTX produced in these lysogens varied from strain to strain and was less than that in PA158.
The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome
Authors: Burman N. , Bergström S. , Restrepo B. I. , Barbour A. G.
Published: October 2006

The relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer‐membrane proteins, known as Vmp proteins. The genes for the variant‐specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2kD) and 364 amino acids (37.1kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome.
A family of dispersed repeats in Mycobacterium leprae
Authors: Woods S. A. , Cole S. T.
Published: October 2006

The genome of the causative agent of leprosy, Mycobacterium leprae, contains at least 28 copies of a dispersed repetitive sequence, RLEP. From nucleotide sequence analysis it was clear that the RLEP element consists of a 545 bp central domain flanked by a 100bp left‐end and a 44bp right‐end, sometimes associated with a 47bp extension. The presence of the left and right ends is variable and this allowed three different RLEP configurations to be defined. When the polymerase chain reaction was used to study variation of the central region at least twelve different classes were detected, suggesting that no two RLEP sequences may be identical. Furthermore, they have few features in common with classical bacterial insertion sequences.
Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence?
Authors: Higgins C. F. , Hinton J. C. D. , Hulton C. S. J. , Owen‐Hughes T. , Pavitt G. D. , Seirafi A.
Published: October 2006

There has been a recent revival of interest in one of the most abundant Escherichia coli proteins, H1 (also called H‐NS). This protein was first identified many years ago as a major component of the bacterial nucleoid, and has been characterized biochemically by several groups. However, no clear function for the protein emerged from these studies. Our thinking has been transformed by recent findings which complement the biochemistry with genetic data. Several mutations, selected over many years by virtue of their diverse effects on gene expression, have turned out to be allelic and to fall within the structural gene for H1. Bringing together the genetics and the biochemistry has demonstrated that the whole is worth more than the sum of the parts! These findings have far‐reaching implications for the mechanisms by which gene expression is regulated and also, perhaps, for the control of bacterial virulence.
Bacillus subtilis levansucrase: amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals
Authors: Petit‐Glatron M. F. , Monteil I. , Benyahia F. , Chambert R.
Published: October 2006

Studies of the equilibrium between native and denatured forms of wild‐type levansucrase showed that the denatured form was predominant at 37°C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal‐dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly‐366→Asp or Gly‐366→Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly‐366→Ser variant was secreted at near‐normal levels and shared a part of the in vitro refolding properties of the wild‐type protein. These differential properties might be related to the ability of the altered region to form a β‐turn structure.

We discuss the possible role of metal ions in the coupling of protein folding and secretion.
Specific binding of VirG to the vir box requires a C‐ terminal domain and exhibits a minimum concentration threshold
Authors: Powei B. S. , Kado C. I.
Published: October 2006

The positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA‐binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989). This paper is an extension of that research and presents evidence on the in vivo DNA‐binding properties of VirG using a transcription interference assay. VirG protein bound specifically to a vir box'response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box. A biphasic response in specific DNA‐binding was observed upon Increasing virG expression, suggesting that specific binding was co‐operatively affected by protein concentration. Certain TrpE′ ‐ ′VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg‐118 of the VirG polypeptide. These data further localize a DNA‐binding domain within VirG, and support a modified model for the regulation of virulence genes in which trans‐ phosphorylation by the coregulator VirA functions to stabilize specific DNA‐binding by low concentrations of VirG, resulting in gene activation. Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987).
Induction of a φC31 prophage inhibits rRNA transcription in Streptomyces coelicolor A3(2)
Authors: Clayton T. M. , Bibb M. J.
Published: October 2006

A lysogen of Streptomyces coelicolor A3(2) containing a thermoinducible mutant of the temperate phage φC31 (φ C31 cts1) was used to obtain synchronous phage development. Filter hybridization experiments indicated a marked reduction in rRNA synthesis after prophage induction. S1 nuclease mapping showed that transcription from each of the four promoters of one rRNA gene set (rrnD) was reduced to approximately the same extent, and that inhibition required protein synthesis. Crude preparations of RNA polymerase from induced lysogens had enhanced transcribing acitivity for φC31 DNA which was lost upon further purification. The purified preparations were unimpaired in their ability to transcribe from the rrnD promoters in vitro and apparently unchanged in poly‐peptide composition. The factor(s) responsible for stimulating phage transcription, and possibly for inhibiting rRNA synthesis, may have been separated from the enzyme during purification.
Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1
Authors: Alm R. A. , Manning P. A.
Published: October 2006

EI Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood.

In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site‐directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid‐exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane‐associated protein.
Surface‐induced swarmer cell differentiation of Vibrio parahaemoiyticus
Authors: McCarter L. , Silverman M.
Published: October 2006

Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface‐induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.