bluuebunny/crossref_metadata_2025 huggingface dataset:
The gene encoding the periplasmic cyclophilin homologue, PPlase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP–CRP complex and negatively regulated by the CytR repressor
Authors: Nørregaard‐Madsen Mads , Myglnd Bente , Pedersen Rolf , Valentin‐Hansen Poui , Søgaard‐Andersen Lotte
Published: October 2006

The rot gene in Escherichia coli encodes PPlase A, a periplasmic peptldyl‐prolyl cis‐trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA‐binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP–CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP–CRP to two sites separated by 53 bp. Moreover, one of the cAMP–CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP–CRP/DNA nucleoprotein complex consisting of the two cAMP–CRP molecules and CytR bound in between. The two regulators bind co‐operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP–CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo‐ and deoxyribonucleoside catabolism or transport.
Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. cartovora in response to DNA‐damaging agents
Authors: Liu Yang , Chatterjee Asita , Chatterjee Arun K.
Published: October 2006

In most soft‐rotting Erwinia spp., including E. carotovora sub sp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (PnI) is activated by DNA‐damaging agents such as mitomycin C (MC). Induction of PnI production in Ecc71 requires a functional recA gene and the rdg locus DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressers of lambdoid phages, specially φ80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix‐turn‐helix motifs. By replacing the rdgB promoter with the IPTG‐inducible tac promoter, we have determined that rdgB by itself can activate PnI production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnIA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of PnI production, are expressed differently in Ecc71.
The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site‐specific recombinease
Authors: Höfte Monica , Dong Qinghan , Kourambas Sophia , Krishnapillai Viji , Sherratt David , Mergeay Max
Published: October 2006

Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site‐specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site‐specific recombination at cer in an E. coii xerC mutant and the E. coii xerC gene could complement a genomic sss mutation in P. aeruginosa.
Expression of the genes coding for the Escherichia coli integration host factor are controlled by growth phase, rpoS, ppGpp and by autoregulation
Authors: Aviv Michal , Giladi Hilla , Schreiber Gideon , Oppenheim Amos B. , Glaser Gad
Published: October 2006

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth‐phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the rel4 and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth‐phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS.

Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF‐binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF‐binding site was found upstream of the himD/hip promoter. AM three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.
Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation
Authors: Ma Charles K. , Koslesnikow Tassia , Rayner John C. , Simons Elizabeth L. , Yim Harry , Simons Robert W.
Published: October 2006

RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS 10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosomebinding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The signficance of these observations for the RNA folding problem in general is discussed.
PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis‐acting sequence upstream of the pilin gene promoter
Authors: Jin Shouguang , Ishimoto Karyn S. , Lory Stephen
Published: October 2006

The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two‐component sensor–regulator family. PilR was overproduced by fusing pilR to the gene for the maltose‐binding protein (malE), yielding a MalE–PilR hybrid protein. The plasmid with the malE–pilR fusion, when introduced into a non‐piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE‐PilR protein was purified from Escherichia coli and used in a series of DNA‐binding studies. A specific pilin promoter‐binding activity of MalE‐PilR was observed in a gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp PilR‐binding site located at the −120 to −80 region, relative to the transcriptional start site of the pilin gene. This PilR‐binding region consists of a nine‐base sequence and three consensus sequences of 5‐(N)4–6C/GTGTC‐3′, in a tandem array in which the first 7–9 bp are bound by the PilR on the non‐Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of sequences complementary to the two middle consensus sequences of the non‐coding strand. A sequence similar to the NifA recognition site (5‐TGT‐(N)11‐ACA‐3′) is also found within the PilR‐binding region. Deletion analysis and disruption of the individual consensus PilR‐binding sequences by site‐directed mutagenesis revealed that all four PilRbinding sites are absolutely required for the PilS/PilR‐mediated pilin gene expression. The presence of four PilR‐binding repeat sequences suggests that PilR protein may bind co‐operatively or as a multimer.
Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae
Authors: Elkins Christopher , Barkley Katherine B. , Carbonetti Nicholas H. , Coimbre Alex J. , Sparling P. Frederick
Published: October 2006

Gonococcal porins (Por) from strains FA19 (Por‐1, serogroup A), MS11 (Por‐2, serogroup B) and FA6434 (Por‐5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non‐denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole‐cell radioimmunoprecipitation and were broadly cross‐reactive within serogroups. Post‐immune, but not pre‐immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outermembrane protein Rmp.
Partial purification of the rat erythrocyte receptor for the channel‐forming toxin aerolysin and reconstitution into planar lipid bilayers
Authors: Gruber Hermann J. , Wilmsen Hans U. , Cowell Simon , Schindler Hansgeorg , Buckley J. Thomas
Published: October 2006

The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N‐glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.
Biosynthesis and functional role of haem O and haem A
Authors: Mogi Tatsushi , Saiki Keitarou , Anraku Yasuhiro
Published: October 2006

Haem O and/or haem A are specifically synthesized for the haem‐copper respiratory oxidases. A 17‐carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen‐reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB‐ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.
The amino‐terminal domain of the P‐pilus adhesin determines receptor specificity
Authors: Haslam David B. , Borén Thomas , Falk Per , Liver Dag , Chou Amy , Xu Zheng , Normark Staffan
Published: October 2006

Pyelonephritic isolates of Escherichia coli commonly express P‐pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P‐pili have been defined, based on varying specificity for galabiose‐containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo‐terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo‐terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino‐terminus of the PapG adhesin and is independent of the complex pilus architecture.
Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA‐binding domain
Authors: Brown David P. , Ganova‐Raeva Lilia , Green Brian D. , Wilkinson Shane R. , Young Michael , Youngman Philip
Published: October 2006

Spo0A is a phosphorylation‐activated transcription factor of Bacillus subtilis. It is a member of the response regulator super‐family of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of speculation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix‐turn‐helix motif, and, therefore, represents the actual DNA‐contacting surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene‐disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spolVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation‐specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A‐like gene in non‐endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A‐like proteins may be exclusive to the endospore‐forming bacteria.
The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation
Authors: Biesen Tim van , Frost Laura S.
Published: October 2006

Most of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F‐like conjugative plasmids. Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system. The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression. Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target. Using electrophoretic mobility shift assays, we have shown that FinO is an RNA‐binding protein that binds to one of the two stem‐loops in FinP and to its complementary structure in traJ mRNA. This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP‐ traJ mRNA duplex fivefold. Thus, TraJ expression appears to be influenced by a unique RNA‐protein interaction that precedes duplex formation between the FinP anti‐sense RNA and its target traJ mRNA.
Multicopy single‐stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli
Authors: Maas Werner K. , Wang Chi , Lima Tania , Zubay Geoffrey , Lim Dongbin
Published: October 2006

Retrons are genetic elements that encode multicopy single‐stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single‐stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem‐loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem‐loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.
High‐frequency S‐layer protein variation in Campylobacter fetus revealed by sapA mutagenesis
Authors: Blaser Martin J. , Wang Enze , Tummuru Murali K. R. , Washburn Ronald , Fujimoto Shuji , Labigne Agnès
Published: October 2006

Campylobacter fetus utilizes paracrystalline surface (S‐) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full‐length S‐layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full‐length S‐layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10‐2. Immunoblots of survivors showed that phenotypic reversion involving high‐level production of full‐length (98, 127 or 149 kDa) S‐layer proteins had occurred. Revertants were serum resistant but caused approximately 10‐fold less bacteraemia in orally challenged mice than did the wild‐type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high‐frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.
The pAMβ1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene
Authors: Chatelier E. Le , Ehrtich S. D. , Jannière L.
Published: October 2006

pAMβ1 is a low‐copy‐number, promiscuous plasmid from Gram‐positive bacteria that replicates by a unidirectional theta‐type mode. Its replication is initiated by an original mechanism, involving the positive rate‐limiting RepE protein. Here we show that the pAMβ1‐encoded CopF protein is involved in negative regulation of the plasmid copy number. CopF represses ‐10‐fold the transcription initiated at the promoter of the repE gene and binds to a 31 bp segment which is located immediately upstream of the ‐35 box of the repE promoter. We propose that CopF inhibits initiation of transcription at the repE promoter by binding to its operator.
The copR gene product of plasmid p1P501 acts as a transcriptional repressor at the essential repR promoter
Authors: Brantl Sabine
Published: October 2006

The amount of the rate‐limiting replication initiator protein RepR of plasmid p1P501 is negatively controlled by an antisense RNA (RNAIII) and a dispensable protein (CopR). Deletions or mutations in either component cause a 10‐20‐fold copy number increase. RNAIII induces transcription attenuation of the repR mRNA: the mode of CopR action remained unclear. To test the function of CopR, transcriptional fusions of promoters pI, pII and pIII with lacZ were integrated into the Bacillus subtilis chromosome. CopR and/or RepR were supplied in trans, and LacZ synthesis measured. The results show that CopR represses the repR promoter pII. Neither CopR nor RepR autoregulate their promoters. Gel mobility shift assays indicate that CopR binds to a 44 bp DNA fragment comprising the inverted repeat upstream of pII.
Early stages in development of the Escherichia coli cell‐division site
Authors: Cook William R. , Rothfield Lawrence I.
Published: October 2006

Development of the Escherichia coli cell division site was studied in wild‐type cells and in non‐septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non‐septate filaments of FtsZ‐ cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild‐type cells and FtsZ‐ filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.
Development of the cell‐division site in FtsA‐filaments
Authors: Cook William R. , Rothfield Lawrence I.
Published: October 2006

Early changes at cell‐division sites were studied in non‐septate filaments induced by growth of ftsATs mutant cells under non‐permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division‐site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.
Identification of an ATP‐binding cassette transport system required for translocation of lipopolysaccharide O‐antigen side‐chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1
Authors: Bronner Dorothea , Clarke Bradley R. , Whitfield Chris
Published: October 2006

The rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D‐galactan I component of the lipopolysaccharide O‐antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP‐binding domain and shares homology with several proteins which function as ATP‐binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane‐spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K‐1 and K‐5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two‐component ABC (ATP‐binding cassette) transporters. E. coli K‐12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O‐antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O‐polysaccharide transport is restored and smooth D‐galactan l‐substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D‐galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane‐spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.
Mode of action of LciA, the lactococcin A immunity protein
Authors: Venema K. , Haverkort R. E. , Abee T. , Haandrikman A. J. , Leenhouts K. J. , de Leij L. , Venema G. , Kok J.
Published: October 2006

Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose‐binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane‐associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A.

The monoclonal antibody specifically reacted with right‐side‐out vesicles obtained from a strain producing the immunity protein. It did not react with inside‐out vesicles of the same strain, or with right‐side‐out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right‐side‐out vesicles obtained from a strain producing only LciA.

The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right‐side‐out vesicles by proteinase K, it is located at the outside of the cell.

The immunity protein contains a putative a‐amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C‐terminal part of the protein, containing the epitope, is on the outside of the cell.

Vesicle‐fusion studies together with leucine‐uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.
High‐frequency homologous plasmid‐plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces
Authors: Xiao J. , Melton R. E. , Kieser T.
Published: October 2006

Non‐transmissible derivatives of the Streptomyces multi‐copy plasmid pIJ101 were mobilized, by co‐integrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low‐copy‐number Streptomyces coeli‐color A3(2) plasmid SCP2*. Efficient co‐integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co‐integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.
Identification of IS 1206, a Corynebacterium glutamicum IS3‐related insertion sequence and phylogenetic analysis
Authors: Bonamy Celine , Labarre Jean , Reyes Oscar , Leblon Gèrard
Published: October 2006

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3‐related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.
glmS of Thermus thermophilus HB8: an essential gene for cell‐wall synthesis identified immediately upstream of the S‐layer gene
Authors: Fernández‐Herrero Luis Angel , Badet‐Denisot Marie‐Ange , Badet Bernard , Berenguer José
Published: October 2006

A 30 kbp chromosomal region containing the S‐layer gene (slpA) from Thermus thermophilus HB8 was cloned from a λ phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604‐amino‐acid protein highly homologous to the glucosamine−6‐P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine−6‐P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coliglmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine−6‐P at 60°C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed diver‐gently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3′ extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their co‐ordinate expression.
The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury‐resistance proteins
Authors: Morby Andrew P. , Hobman Jon L. , Brown Nigel L.
Published: October 2006

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501 was replaced by serine, and the mercury‐resistance phenotypes of the mutants were determined in Escherichia coli. Cys−24 and Cys−25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys−76‐Ser, Cys−82‐Ser or Gly−38‐Asp in MerT or Cys−36‐Ser in MerP all reduced transport and resistance. Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys−33‐Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deleting merP on mutations in merT were tested. The 116‐amino‐acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane.
Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning
Authors: Mojica F.J.M. , Ferrer C. , Juez G. , Rodríguez‐Valera F.
Published: October 2006

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33–39 bp unique sequences. This structure extends for long stretches — 1.4kb at one location in H. mediterranei chromosome and about 3kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.
Cloning and characterization of GPD2, a second gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1
Authors: Eriksson Peter , André Lars , Ansell Ricky , Blomberg Anders , Adler Lennart
Published: October 2006

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino‐terminal extension of unknown function. GPD2 is located on chromosome VII and cross‐hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild‐type cells. Analysis of gene transcription by a CAT‐reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on non‐fermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8–17‐fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was it induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4M NaCl.
tRNA genes and pathogenicity islands: influence on virulence and metabolic properties of uropathogenic Escherichia coli
Authors: Ritter Angelika , Blum Gabriele , Emödy Levente , Kerenyi Monika , Böck August , Neuhieri Bernhard , Rabsch Wolfgang , Scheutz Flemming , Hacker Jörg
Published: October 2006

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine‐specific tRNA locus leuX. Mutant strain 536−21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536−21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hly I, hly II) and P‐related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition to in vivo virulence are strongly dependent on the intact tRNA loci selC and leuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genes seIC and leuX were cloned from the genome of strain 536 and then introduced into the mutant 536−21. Our results clearly show that the selenocysteine‐specific tRNA (tRNASec) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine‐specific tRNA5Leu, encoded by leuX, influences a number of properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for full in vivo virulence. While the tRNASec is directly involved in the production of FDHs, the leuX specific tRNA5Leu appears to influence the expression of various factors through specific transcriptional or translational control mechanisms.
SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion
Authors: Benjelloun‐Touimi Zineb , Sansonetti Philippe J. , Parsot Claude
Published: October 2006

In addition to lpa proteins and lcsA, which are involved in entry into epithelial cells and intercellular spread, respectively, Shigella secretes a 110 kDa protein, designated SepA. We report the identification, cloning, and nucleotide sequence determination of the sepA gene, analysis of SepA secretion, and construction and characterization of a sepA mutant. The sepA gene is carried by the virulence plasmid and codes for a 150 kDa precursor. Upon secretion, which does not involve accessory proteins encoded by the virulence plasmid, the precursor is converted to a mature protein of 110 kDa by two cleavages removing an N‐terminal signal sequence and a C‐terminal fragment. Extensive similarities were detected between the sequence of the first 500 residues of mature SepA and the N‐terminal region of lgA1 proteases from Neisseria gonorrhoeae and Haemophilus influenzae, the Tsh haemagglutinin of an avian pathogenic Escherichia coli, and the Hap protein involved in adhesion and penetration of H. influenzae. The C‐terminal domain of the SepA precursor, which is not present in the secreted protein, exhibits sequence similarity with pertactin of Bordetella pertussis and the ring‐forming protein of Helicobacter mustelae. Construction and phenotypic characterization of a sepA mutant indicated that SepA is required neither for entry into cultured epithelial cells nor for intercellular dissemination. However, in the rabbit ligated ileal loop model, the sepA mutant exhibited an attenuated virulence, which suggests that SepA might play a role in tissue invasion.
Characterization of a novel fibronectin‐binding surface protein in group A streptococci
Authors: Kreikemeyer B. , Talay S.R. , Chhatwal G.S.
Published: October 2006

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin‐binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin‐binding protein of S. pyogenes, designated SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa protein exhibiting features of membrane‐anchored surface proteins of Gram‐positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin‐binding domain within the C‐terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin‐binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross‐reactivity could be detected between anti‐Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.
PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion
Authors: Pegues David A. , Hantman Michael J. , Behlau Irmgard , Miller Samuel I.
Published: October 2006

Previously, the PhoP‐repressed locus prgH was identified as important for signalling epithelial cells to endocytose Salmonella typhimurium. Characterization of prgH revealed that it is an operon of four genes encoding polypeptides of 392 (prgH), 80 (prgl), 101 (prgJ) and 252 amino acid residues (prgK). Synthesis of the 2.6 kb prgHIJK transcript was repressed in bacteria that activate PhoP/PhoQ, indicating that PhoP/PhoQ regulates prgHIJK by transcriptional repression. The prgl, prgJ and prgK predicted gene products were similar to Shigella flexneri and Yersinia enterocolitica proteins required for secretion of lpa and Yop virulence factors. Analysis of the culture supernatants from wild‐type S. typhimurium demonstrated that at least 25 polypeptides larger than 14 kDa could be detected. In contrast, prgH1::TnphoA, phoP‐constitutive and hil‐deletion mutants had significant defects in their supernatant protein profiles. The invasion and supernatant protein profile defects of the prgH1::TnphoA mutant were both complemented by a 5.1 kb plasmid that included prgHIJK. These results suggest that PhoP/PhoQ regulates extracellular transport of proteins by transcriptional repression of secretion determinants and that secreted proteins may be involved in signalling epithelial cells to endocytose bacteria.
A multiple site‐specific DNA‐inversion model for the control of Ompi phase and antigenic variation in Dichelobacter nodosus
Authors: Moses Eric K. , Good Robert T. , Sinistaj Meri , Billington Stephen J. , Langford Christopher J. , I. Rood Julian
Published: October 2006

The molecular cloning and sequence analysis of four structurally variant linked genes (omp1A,B,C,D) that encode the major outer membrane protein of Dichelobacter nodosus strain VCS1001 are described. The isolation of rearranged copies of omp1A and omp1B, and the identification in the 5′ regions of all four genes of short cross‐over‐site sequences that were similar to the Din family of cross‐over‐site sequences, suggested that site‐specific DNA inversion was involved in omp1 rearrangement. Evidence for site‐specific inversion of the 497 bp DNA fragment, which was located between the divergently orientated omp1A and omp1B genes, and which contained the promoter and 5t' coding sequence of omp1, was obtained by polymerase chain reaction‐mediated amplification of inverted forms of these genes. However, to account for all of the omp1 gene copies cloned in this study, a more widespread inversion phenomenon must be involved in the rearrangement of these genes and a model for multiple site‐specific DNA inversions at the omp1 locus is described. In this model the four structurally variant omp1 genes can be assembled from one of four structurally variant C‐terminal coding regions and a conserved N‐terminal coding region and can be expressed from a single promoter. It is postulated that this genetic capability endows D. nodosus with the ability to switch the antigenic specificity of one of its major surface proteins.
Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid
Authors: Actis Luis A. , Tolmasky Marcelo E. , Crosa Lidia M. , Crosa Jorge H.
Published: October 2006

The pJM1‐encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy‐terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa pro‐FatB precursor protein.
Programmed cell death in bacteria: proteic plasmid stabilization systems
Authors: Jensen Rasmus Bugge , Gerdes Kenn
Published: October 2006

Bacterial plasmids are stabilized by a number of different mechanisms. Here we describe the molecular aspects of a group of plasmid‐encoded gene systems called the proteic killer gene systems. These systems mediate plasmid maintenance by selectively killing plasmid‐free cells (post‐segregational killing or plasmid addiction). The group includes ccd of F, parD/pem of R1/R100, parDE of RP4/RK2, and phd/doc of P1. All of these systems encode a stable toxin and an unstable antidote. The antidotes prevent the lethal action of their cognate toxins by forming tight complexes with them. The antidotes are degraded by cellular proteases. Thus, the different decay rates of the toxins and antidotes seem to be the molecular basis of toxin activation in plasmid‐free cells. The operons encoding the toxins and antidotes are autoregulated at the level of transcription either by a complex formed by the toxins and the cognate antidotes or by the antidote alone. The cellular targets of the killer proteins have been determined to be DNA gyrase in the case of ccd of F and DnaB in the case of parD of R1. Surprisingly, the Escherichia coli chromosome encodes at least two of these peculiar gene systems.