bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1
Authors: Jensen Rasmus Bugge , Grohmann Elisabeth , Schwab Helmut , Díaz‐Orejas Ramón , Gerdes Kenn
Published: October 2006

A number of plasmid‐encoded gene systems are thought to stabilize plasmids by killing plasmid‐free cells (also termed post‐segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication‐arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non‐metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady‐state loss rates and the effect on cell proliferation in the plasmid replication‐arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid‐free segregants, whereas parD seems to act by inhibiting cell division of plasmid‐free segregants.
Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2)
Authors: Kelemen Gabriella H. , Plaskitt Kitty A. , Lewis Cinzia G. , Findlay Kim C. , Buttner Mark J.
Published: October 2006

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co‐transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.
Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading
Authors: Uchiya Kei‐ichi , Tobe Toru , Komatsu Keiko , Suzuki Toshihiko , Watarai Masahisa , Fukuda Ichirou , Yoshikawa Masanosuke , Sasakawa Chihiro
Published: October 2006

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild‐type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus‐plaque‐forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.
Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP
Authors: Tagami Hideaki , Inada Toshifumi , Kunimura Tadashi , Aiba Hiroji
Published: October 2006

CRP—cAMP‐dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second‐site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP‐independent catabolite repression has been a long‐standing mystery. Here we address the question of how glucose inhibits the expression of β‐galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of β‐galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS—PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with β‐galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya− crp* cells and that the autoregulatory circuit of the crp gene is involved in the down‐regulation of CRP* expression by glucose.
Evidence that the transcriptional activator Spo0A interacts with two sigma factors in Bacillus subtilis
Authors: Baldus Jean M. , Buckner Cindy M. , Moran Charles P.
Published: October 2006

The transcriptional regulator Spo0A activates transcription from two types of promoters. One type of promoter is used by RNA polymerase containing τA, whereas the other type is used by RNA polymerase containing τH. There are Spo0A‐binding sites near the −35 region of both types of promoters. It has been reported that some transcriptional regulators that bind near the −35 regions of promoters directly interact with the sigma subunit of RNA polymerase. Therefore, we looked for evidence that Spo0A interacts with both sigma factors by searching for single amino acid substitutions in these factors that specifically prevent expression from Spo0A‐dependent promoters, but that do not decrease activity of Spo0A‐independent promoters. Two such amino acid substitutions were isolated in τA and one was isolated in τH. The amino acid substitutions in τA prevented expression from the Spo0A‐activated promoters, spoIIG and spoIIE, but expression was not impaired from the Spo0A‐independent, τA‐dependent promoter tms or from the Spo0A‐activated, τH‐dependent promoter, spoIIA. The amino acid substitution in τH prevented expression from the spoIIA promoter but not from the Spo0A‐independent promoter, citGp2, which is used by τH‐RNA polymerase. All of these amino acid substitutions occur in the carboxyl terminus of the sigma factors. These amino acid substitutions may define the sites of contact between the sigma factors and Spo0A. The ability of response regulators such as Spo0A to interact with multiple sigma factors may increase the variety of responses made by bacteria using a limited number of transcription factors.
A transcription terminator signal necessary for plasmid Collb‐P9 replication
Authors: Mori Atsushi , Ito Koichi , Mizobuchi Kiyoshi , Nakamura Yoshikazu
Published: October 2006

Replication of the Inclα plasmid CoIIb‐P9 requires the repZ gene, which encodes an essential, unstable initiator protein termed RepZ. Although many functional features of the CoIIb‐P9 replicon resemble those of structurally unrelated IncFII plasmids R1 and NR1, the role of transcription of repZ towards the replication origin is poorly understood. Using a series of deletion and substitution mutants of the CoIIb‐P9 replicon, we found that RepZ prefers to act in cis and that a spacer sequence between repZ and the origin is required for replication. This spacer element, referred to as CIS, retained strong transcription terminator activity. Efficient transcription terminators, whether Rho‐dependent or ‐independent, were capable of replacing CIS function for in vivo replication; CoIIb‐P9 replicated better as transcription terminated more efficiently within CIS. When the CIS element was substituted for by a strong Rho‐dependent terminator, such as λ tR1 or E. coli trp tt', in vivo replication of these recombinant replicons became dependent on the Rho factor, in contrast to the authentic CoIIb‐P9 replicon.
Leucine‐responsive regulatory protein plays dual roles as both an activator and a repressor of the Escherichia coli pap fimbrial operon
Authors: Van Der Woude M. W. , Kaltenbach L.S. , Low D.A.
Published: October 2006

The expression of the pap pilus operon of Escherichia coli is under a phase‐variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states. In this study, we explore the roles of leucine‐responsive regulatory protein (Lrp) and the histone‐like protein H‐NS in the regulation of pap phase variation. Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H‐NS, each of which can act independently as transcriptional repressors. Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H‐NS does not. In contrast, in the ON state, Lrp, in conjunction with Papl, activates pap transcription. This activation is not merely a result of alleviating the H‐NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H‐NS and Lrp. Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus. Together, our results show that Lrp functions as a transcriptional activator in phase‐ON cells and as a repressor of basal transcription in phase‐OFF cells. Because pap phase variation occurs in the absence of H‐NS, it is not clear what role this regulatory protein plays in pap gene regulation.
Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1
Authors: Latifi Amel , Winson Michael K. , Foglino Maryline , Bycroft Barrie W. , Stewart Gordon S. A. B. , Lazdunski Andrée , Williams Paul
Published: October 2006

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the autoinducer molecule N(3‐oxododecanoyl)‐l‐homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase‐negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N(3‐oxohexanoyl)‐l‐homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of IasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re‐established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum‐sensing modulons interactively regulate gene expression in P. aeruginosa.
Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N‐acylhomoserine lactone signal molecules
Authors: Throup John P. , Camara Miguel , Briggs Geoffrey S. , Winson Michael. K. , Chhabra Siri Ram , Bycroft Barrie W. , Williams Paul , Stewart Gordon S.A.B.
Published: October 2006

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high‐performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N‐hexanoyl‐l‐homoserine lactone (HHL) and N‐(3‐oxohexanoyl)‐l‐homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with ≈20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5′ transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N‐acylhomo‐serine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two‐dimensional SDS‐PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.
Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides
Authors: Ward Mandy J. , Bell Adam W. , Hamblin Paul A. , Packer Helen L. , Armitage Judith P.
Published: October 2006

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8‐N using a probe based on the 3′ terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA, cheW, and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl‐accepting chemotaxis protein (MCP)‐dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild‐type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over‐expressed in E. coli. The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP‐independent sensing. The MCP‐dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.
The leucine zipper of Aeromonas salmonicida AbcA is required for the transcriptional activation of the P2 promoter of the surface‐layer structural gene, vapA, in Escherichia coli
Authors: Noonan Brian , Trust Trevor J.
Published: October 2006

The Aeromonas salmonicida AbcA protein is involved in the synthesis of the O‐polysaccharide side‐chains on the lipopolysaccharide and is also capable of enhancing the expression of the structural gene for the A‐layer, vapA, when cloned into Escherichia coli. The P2 promoter of the vapA gene of A. salmonicida was cloned into a promoter probe vector and expression in E. coli was monitored. The expression of P2::lacZ was shown to be increased when abcA was provided in trans. AbcA contains an N‐terminal ATP‐binding domain as well as a C‐terminal leucine zipper domain. Site‐directed mutagenesis has been used to show that the ATP‐binding domain is required for the synthesis of the O‐polysaccharide side‐chains, but not for the enhancement of vapA expression. Conversely, the leucine zipper is needed for the increase in vapA expression, but not for O‐polysaccharide side‐chain synthesis. This indicates that AbcA is a bifunctional protein that can influence the synthesis of the two principle antigenic components of the A. salmonicida cell surface.
NodS is an S‐adenosyl‐l‐methionine‐dependent methyltransferase that methylates chitooligosaccharides deacetylated at the non‐reducing end
Authors: Geelen Danny , Leyman Barbara , Mergaert Peter , Klarskov Klaus , Van Montagu Marc , Geremia Roberto , Holsters Marcelle
Published: October 2006

In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain‐specific substitutions. In Azorhizobium caulinodans strain ORS571 these modifications are an O‐arabinosyl group, an O‐carbamoyl group, and an N‐methyl group. Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors. Previously we have shown that NodS is an S‐adenosyl‐l‐methionine (SAM)‐binding protein, essential for the l‐[3H‐methyl]‐methionine labelling of ORS571 Nod factors in vivo. Here, we present an in vitro assay showing that NodS from either A. caulinodans or Rhizobium species NGR234 methylates end‐deacetylated chitooligosaccharides, using [3H‐methyl]‐SAM as a methyl donor. The enzymatic and SAM‐binding activity were correlated with the nodS gene and localized within the soluble protein fraction. The A. caulinodans nodS gene was expressed in Escherichia coli and a glutathione‐S‐transferase—NodS fusion protein purified. This protein bound SAM and could methylate end‐deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo‐chitooligosaccharides. These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides.
RNase P — a ‘Scarlet Pimpernel’
Authors: Kirsebom Leif A.
Published: October 2006

RNase P is responsible for the maturation of the 5′‐termini of tRNA molecules in all cells studied to date. This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors. This review covers what is currently known about the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage‐site selection. Recent genetic and biochemical data show that the two C residues in the 3′‐terminal CCA sequence of a precursor interact with the enzyme through Watson‐Crick base‐pairing. This is suggested to result in unfolding of the amino acid acceptor‐stem and exposure of the cleavage site. Furthermore, other close contact points between M1 RNA and its substrate have recently been identified. These data, together with the two existing three‐dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by this elusive enzyme.
Why does Escherichia coli recycle its cell wall peptides?
Authors: Park J.T.
Published: October 2006

This review discusses the phenomenon of recycling of cell wall peptides. It is a major, though non‐essential, pathway of the cell and is required for induction of β‐lactamase. Consequently, the recycling pathway is viewed as a possible signalling vehicle, informing the cell of the condition of the murein sacculus, an essential structure existing outside the cell itself. As the study of this phenomenon is in its infancy, several speculations are offered for a possible regulatory function.
Maturation pathway of nisin and other lantibiotics: post‐translationally modified antimicrobial peptides exported by Gram‐positive bacteria
Authors: De Vos Willem M. , Kuipers Oscar P. , Van Der Meer Jan Roelof , Siezen Roland J.
Published: October 2006

Lantibiotics form a family of highly modified peptides which are secreted by several Gram‐positive bacteria. They exhibit antimicrobial activity, mainly against other Gram‐positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self‐protection proteins, processing enzymes and regulatory proteins have been identified. This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food‐grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry.
An intron‐containing meiosis‐induced recombination gene, rec15, of Schizosaccharomyces pombe
Authors: Lin Yukang , Smith Gerald R.
Published: October 2006

Mutations in the rec15 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies, in the three intervals tested, as much as 1000‐fold but have no detectable mitotic phenotype. The rec15 gene was mapped to within 1 cM of mat1. The gene was cloned by genetic complementation and its nucleotide sequence determined. Deletion analysis and gene replacement confirmed that the clones contained the rec15 gene on DNA fragments as short as 1.3 kb. The nucleotide sequence of the 1.3 kb fragment predicted that rec15 had a 49 bp intron separating two exons encoding a 180‐amino‐acid polypeptide product. This predicted intron was confirmed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis. During thermally induced meiosis in a pat1–114 (Ts) mutant, the rec15 transcripts were induced to maximal levels at 2–3 h but were present at much lower levels before and after this time. The transient induction of the transcripts and the phenotype of a rec15 null (deletion) mutation suggest that the rec15 gene product is required during the early stages of meiosis for meiotic recombination.
The extreme C‐terminus is required for secretion of both the native polygalacturonase (PehA) and PehA‐Bla hybrid proteins in Erwinia carotovora subsp. carotovora
Authors: Palomäki T. , T. Saarilahti H.
Published: October 2006

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic β‐lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out‐terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA‐Bla hybrid proteins and PehA variants lacking at least four aa from the C‐terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site‐directed mutagenesis of the codons −4 and −3 (encoding Asn‐373 and Val‐374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val‐374 was important both for the structure and secretion of PehA, while the Asn‐373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C‐terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C‐terminal extremity of a secreted molecule; and (iii) residues within the C‐terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.
MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination
Authors: Allaoui Abdelmounaaïm , Sansonetti Philippe J. , Ménard Robert , Barzu Simona , Mounier Joelle , Phalipon Armelle , Parsot Claude
Published: October 2006

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the lpa proteins, and their dedicated secretion apparatus, the Mxi—Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi‐Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild‐type copy of mxiG (which restored lpa secretion, entry into HeLa cells, and cell‐to‐cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect lpa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi‐Spa translocon are involved not only in entry but also in spread of shigella between epithelial cells.
Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine‐mediated repression, but still able to activate atσ70 promoters
Authors: Kwok Terry , Yang Ji , Pittard A.J. , Wilson Timothy J. , Davidson Barrie E.
Published: October 2006

In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates σ70, not σ54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine‐mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine‐ and phenylalanine‐mediated repression of aroP. In contrast, it was able to effect wild‐type levels of phenylalanine‐mediated repression of aroG, tryptophan‐mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine‐dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine‐mediated repression by TyrR but not for tyrosine‐ or phenylalanine‐mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP‐dependent aromatic amino acid‐binding site.
Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds
Authors: Shingler V. , Pavel H.
Published: October 2006

The NtrC‐like regulator DmpR controls transcription from the dmp operon that encodes the enzymes for catabolism of phenol and some related aromatic compounds. DmpR activates transcription from the σ54‐dependent dmp‐operon promoter in the presence of pathway substrates or structural analogues in the growth medium. Using affinity‐purified DmpR and a truncated derivative, we show here that aromatic compounds directly activate the ATPase activity of this protein in vitro, and that the amino‐terminal domain represses this activity in the absence of an aromatic ligand. In order to dissect the activation process, derivatives of DmpR exhibiting single amino acid changes were isolated and their effector‐dependence and specificity profiles were analysed in vivo. The mechanistic implications of the phenotypes of these mutants are discussed.
Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme
Authors: Venema Koen , Kok Jan , Marugg Joey D. , Toonen Marjolein Y. , Ledeboer Aat M. , Venema Gerard , Chikindas Michael L.
Published: October 2006

The bacteriocin pediocin PA‐1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA‐1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N‐terminal domains of PedD and a subgroup of bacteriocin ABC‐transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.
Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens
Authors: Sezonov Guennadi , Hagège Juliette , Pernodet Jean‐Luc , Friedmann Annick , Guérineau Michel
Published: October 2006

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2 which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.
A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis
Authors: Skurnik Mikael , Venho Reija , Toivanen Paavo , Al‐Hendy Ayman
Published: October 2006

Yersinia enterocolitica serotype O:3 strain 6471/76‐c (YeO3‐c) was sensitive to bacteriophage φR1‐37 when grown at 37°C but not when grown at 22°C because of steric hindrance by abundant lipopolysaccharide (LPS) O‐side chain (O‐antigen) expressed at 22°C. The transposon library of YeO3‐c was grown at 37°C and screened for phage φR1‐37‐resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O‐antigen (LPS‐smooth), (ii) phage receptor mutants not expressing O‐antigen (LPS‐rough), and (iii) LPS‐smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the trs operon is involved in the biosynthesis of the LPS outer core in YeO3‐c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarity to known polysaccharide biosynthetic genes of various Gram‐negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus; (ii) the biosynthesis of the core of YeO3‐c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage φR1‐37 receptor structures; (iv) the homopolymeric O‐antigen of YeO3‐c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk—hemH and galE—gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage φR1‐37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.
Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus
Authors: Collazo Carmen M. , Zierler Michael K. , Gatan Jorge E.
Published: October 2006

We have carried out a functional analysis of invl and invJ, two Saimonella typhimurium genes required for this organism to gain access So cultured mammalian cells. These genes are located immediately downstream of invC, a previously identified gene also required for bacterial invasion. Non‐polar mutations in either of these genes rendered S. typhimurium severely defective for entry into cultured epithelial cells, although these mutations did not affect the ability of these organisms to attach to those cells. Nucleotide sequence analysis revealed that the invl and invJ genes encode proteins with molecular weights of 18077 and 36415, respectively. Polypeptides of similar sizes were observed when these genes were expressed in a bacteriophage T7 RNA polymerase‐based expression system. Comparison of the predicted sequences of Invl and InvJ with translated sequences in the existing databases indicated that these proteins are Identical to the previously identified S. typhimurium SpaM and SpaN proteins. Further analysis of these sequences revealed regions of homology between Invl and the N‐terminus of lpaB of Shigella spp. and between InvJ and EaeB of enteropathogenic Escherichia coli. Localization studies by immunoblot analysis indicated that InvJ is secreted to the culture supernatant, a surprising finding since this protein also lacks a typical signal sequence. Mutations in invG and inyC, two members of the Salmonella inv locus, effectively prevented the transport of InvJ to the culture supernatant. Thus, InvJ is the first identified target of the protein secretion apparatus encoded in the inv locus and therefore a candidate to have effector functions related to bacterial entry.
A small gene, designated comS, located within the coding region of the fourth amino acid‐activation domain of srfA, is required for competence development in Bacillus subtilis
Authors: Hamoen Leendert W. , Eshuis Hans , Jongbloed Jan , Venema Gerard , van Sinderen Douwe
Published: October 2006

The valine‐activation domain‐encoding portion of the srfA locus (srfA‐d4) is not only involved in the non‐ribosomal synthesis of surfactin, but is also required for the regulation of competence development. In this study we show that impairment of the adenylation activity of the valine‐activating domain did not affect competence development. Deletion analysis and complementation studies delineated the competence‐required portion of srfA‐d4 to a 168bp fragment, which contains a small open reading frame (ORF), designated comS, encoding a polypeptide of 46 amino acids, embedded within, but translated in, a frame different from that of srfA‐d4. Introduction of an amber mutation in the comS‐coding frame prevented competence development, demonstrating the involvement of comS in this prokaryotic specialization process.
Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial ceils
Authors: Geme Joseph W. St. , Cutter David
Published: October 2006

Hemophilus influenzae type b is a Gram‐negative bacillus that initiates infection by colonizing the upper respiratory tract. Previous studies have established that H. influenzae haemagglutinating pili possess adhesive properties and influence the process of colonization. Additional studies suggest the presence of a second H. influenzae adhesin distinct from haemagglulinating pili. In the present study, we examined a non‐piliated H. influenzae type b strain by transmission electron microscopy and visualized occasional short, thin, surface fibrils. Subsequently, we isolated a spontaneous mutant that lacked surface fibrils and was deficient in adherence to cultured human epithelial cells. Using a cloning strategy that exploited this mutant, we isolated a fragment of DNA that promotes in vitro adherence to human epithelial cells when expressed in laboratory strains of Escherichia coli. Mutagenesis of this fragment in a series of H. influenzae type b strains resulted in loss of expression of surface fibrils and a marked decrease in attachment. Furthermore, restoration of surface fibril expression was associated with reacquisition of wild‐type levels of adherence. Our results are consistent with the conclusion that H. influenzae type b surface fibrils have adhesive capacity. We speculate that these organelles facilitate colonization of the human respiratory tract.
A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1 is essential for virulence: histamine is a precursor in the biosynthesis of anguibactin
Authors: Tolmasky Marcelo E. , Actis Luis A. , Crosa Jorge H.
Published: October 2006

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJMl which encodes a histidine decarboxlase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44 259.69 Da. The amino acid sequence has extensive homology with the pyridoxal‐P‐dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3‐HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJMI Iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron‐limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore. immunoblot analysis of production of β‐galactosidase by V. anguillarum strains carrying the lacZ fusions demonstrated that expression of histidine decarboxylase is not regulated by the iron concentration of the medium.
Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P‐type ATPase and a cation‐binding protein associated with copper transport
Authors: Ge Zhongming , Hiratsuka Koji , Taylor Diane E.
Published: October 2006

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, oRF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611‐amino‐acid sequence of ORF1, a P‐type ATPase (designated hpCopA), had striking homology (29‐38%) with several bacterial P‐type ATPases and contained the potential functional domains conserved in P‐type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion‐transporting protein, and contains the heavy metal‐binding motif. Disruption of ORF1 with a chloramphenicol‐resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alternation of susceptibility to Ni2+, Cd2+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation‐transporting system which is associated with copper export out of the H. pylori cells.
Region II of the Haemophilus influenzae Type b capsulation locus as involved in serotype‐specific polysaccharide synthesis
Authors: Van Eldere Johan , Brophy Lisa , Loynds Barbara , Celts Patrick , Hancock Ian , Carman Stephen , Kroll J. Simon , Moxon E. Richard
Published: October 2006

The central (serotype‐specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP‐ribitolpyrophosphorylase. We present evidence that growth of polysaccharide chains takes place through the alternating addition of single sugar nucleotides.
Regulation of citrate‐dependent iron transport of Escherichia coli: FecR is required for transcription activation by Feel
Authors: Ochs Martina , Veitinger Sabine , Kim InSook , Weiz Dietrich , Angerer Annemarie , Braun Volkmar
Published: October 2006

Citrate‐dependent Fe3+ transport into Escherichia coli K‐12 is induced by iron and citrate. The inducer is probably ferric dicitrate which does not have to be taken up into the cytoplasm to induce transcription of the fec transport genes. Two regulatory genes, fed and fecR, located upstream of the fecABCDE transport genes, are required for induction. We report that in vivo the chromosomally encoded Feel protein activates transcription of the fecA and fecB transport genes in response to ferric citrate and the FecR protein. Cells expressing chromosomally and plasmid‐encoded truncated FecR derivatives no longer responded to ferric citrate and expressed the fec transport genes constitutively. The smallest active FecR derivative contained 59 amino acid residues as compared to the 317 residues of wild‐type FecR. Constitutive induction was lower than induction of the FecR wild‐type strain by ferric citrate. It is concluded that the N‐terminal portion of FecR activates Feel and that the C‐terminal portion of FecR responds to ferric citrate. Transcription of the fee transport genes is positively regulated by Feel and FecR and negatively regulated by the Fe2+‐Fur repressor. Transcription activation and repression may occur independently of each other.
Reinitiation kinetics in eight dnaA(Ts) mutants of Escherichia coli: rifampicin‐resistant Initiation of chromosome replication
Authors: Hansen Flemming G.
Published: October 2006

The kinetics of reinitiation of chromosome replication of eight dnaA(Ts) mutants was investigated in an isogenic set of strains. Five mutants (167, 46, 601, 606 and 5) are classified as reversible, since they can reinitiate at 30 C without protein synthesis, whereas the other three (508, 205, 204) require protein synthesis. In the presence of protein synthesis, reversible mutants initiate one round of replication rapidly after a shift to 30δC, indicating that they contain active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15–20min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42δC and only require synthesis of small amounts of new DnaA protein before initiation at 30δC, whereas dnaA205 accumulates DnaA protein for some time at 30δC before reaching the initiation threshold. Three of the reversible mutants (5, 601, and 606) exhibited, in addition to the protein synthesis‐independent initiation capacity, an RNA synthesis‐independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein, strongly suggesting that mutant DnaA protein is responsible for both.
Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(null) rhn mutant of Escherichia coli
Authors: Hansen Flemming G. , Atlung Tove
Published: October 2006

The kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(null) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild‐type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild‐type concentration (24 ng of DnaA protein per ml cells at OD450= 1.0) At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.
Activation and repression of the yeast ARO3 gene by global transcription factors
Authors: Künzler Markus , Springer Christoph , Braus Gerhard H.
Published: October 2006

The ARO3 gene of Saccharomyces cerevisiae codes for the phenylalanine‐inhibited 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate synthase (EC 4.1.2.15) and is regulated by the general control system of amino acid biosynthesis through a single GCN4‐binding site in its promoter. A combined deletion and mutation analysis of the ARO3 promoter region in a δgcn4‐background revealed two additional regulatory systems involved in ARO3 transcription. The ARO3 gene is (i) activated through a sequence element which binds the multifunctional DNA‐binding protein ABF1 in vitro and (ii) repressed through an URS1 element, which binds the same protein in vitro as the URS1 element In the CAR1 promoter. Since both the ABF1‐binding site and the URS1 element represent cis‐acting elements of global transcription regulatory systems in yeast, the ARO3 gene is the first example of a GCN4‐regulated gene which is both activated and repressed by global transcription factors. Activation of the ARO3 gene through the ABF1‐binding site and repression through the URS1 element seem to be independent of each other and independent of activation by the GCN4 protein.