bluuebunny/crossref_metadata_2025 huggingface dataset:
The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane‐localized transcriptional activators on a single promoter
Authors: Krukonis Eric S. , Yu Rosa R. , DiRita Victor J.
Published: March 2003

ToxR is required in Vibrio cholerae for transcriptional activation of the toxT gene, the protein product of which activates numerous genes involved in virulence. Although ToxR cannot activate the toxT promoter in Escherichia coli, the products of the tcpPH operon are shown here to activate the toxT promoter, and co‐expression with ToxRS enhances activation. An identical pattern was seen in a ΔtcpPΔtoxR strain of V. cholerae when TcpPH or ToxRS was expressed from plasmids. Although overexpression of the TcpP/H proteins in V. cholerae partially complemented both a ΔtoxR strain and a ΔtcpPΔtoxR double mutant for toxin production and toxT–lacZ activation, the presence of ToxR greatly increased their expression. Analysis of a toxT–lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR binding site. This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, which showed that TcpP interacts with the promoter region from −51 to −32, whereas ToxR protected a region from −100 to −69. In addition, membranes containing endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP. Characterization of a number of tcpP substitution mutants revealed one derivative (TcpP‐H93L) that, when overexpressed, was markedly defective for toxT activation, cholera toxin and TcpA (toxin co‐regulated pilus) production and DNA binding; however, toxT activation by TcpP‐H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation. Two additional mutant derivatives, TcpP‐W68L and TcpP‐R86A, failed to activate toxT or direct toxin and TcpA production in the presence or absence of ToxR. Both TcpP‐W68L and TcpP‐R86A, like TcpP‐H93L, were defective for DNA binding. Finally, a ToxR mutant derivative, ToxR‐G80S, served to separate the different roles of ToxR on different promoters. Although ToxR‐G80S was inefficient at activating the ompU promoter in V. cholerae (ompU encodes an outer membrane porin regulated by ToxR), it was fully capable of activating the toxT promoter. These data suggest that ToxR is not a direct activator in the toxT expression system but, instead, enhances the activity of TcpP, perhaps by recruiting it to the toxT promoter under conditions in which expression levels of TcpP are too low for it to activate toxT efficiently on its own.
Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci
Authors: Svensson Mikael D. , Scaramuzzino Dominick A. , Sjöbring Ulf , Olsén Arne , Frank Charlotte , Bessen Debra E.
Published: March 2003

Primary infection of the human host by group A streptococci (GAS) most often involves either the epidermis of the skin or the oropharyngeal mucosa. A humanized in vivo model for impetigo was used to investigate the basis for host tissue tropism among GAS. Disruption of the speB gene (encoding for a secreted cysteine proteinase) led to a loss of virulence for two impetigo‐derived strains (M‐types 33 and 53), as evidenced by a diminution in tissue damage and a lack of reproductive growth. The level of cysteine proteinase activity in overnight cultures was associated with the extent of gross pathological changes induced by strains displaying varied degrees of virulence in the impetigo model. Moreover, high levels of secreted cysteine proteinase activity correlated with a genetic marker for preferred tissue site of infection at the skin (emm pattern D). The addition of exogenous SpeB to a speB mutant (emm pattern D) or to an avirulent throat‐like strain (emm pattern A) led to increased bacterial reproduction at the skin. The data provide both experimental and epidemiological evidence for a critical role of a secreted bacterial protease in promoting host tissue‐specific infection.
Genetic basis for the β‐haemolytic/cytolytic activity of group B Streptococcus
Authors: Pritzlaff Craig A. , Chang Jennifer C. W. , Kuo Shrin P. , Tamura Glen S. , Rubens Craig E. , Nizet Victor
Published: March 2003

Group B streptococci (GBS) express a β‐haemolysin/cytolysin that contributes to disease pathogenesis. We report an independent discovery and extension of a genetic locus encoding the GBS β‐haemolysin/cytolysin activity. A plasmid library of GBS chromosomal DNA was cloned into Escherichia coli, and a transformant was identified as β‐haemolytic on blood agar. The purified plasmid contained a 4046 bp insert of GBS DNA encoding two complete open reading frames (ORFs). A partial upstream ORF (cylB) and the first complete ORF (cylE) represent the 3′ end of a newly reported genetic locus (cyl) required for GBS haemolysin/cytolysin activity. ORF cylE is predicted to encode a 78.3 kDa protein without GenBank homologies. The GBS DNA fragment also includes a previously unreported ORF, cylF, with homology to bacterial aminomethyltransferases, and the 5′ end of cylH, with homology to 3‐ketoacyl‐ACP synthases. Southern analysis demonstrated that the cyl locus was conserved among GBS of all common serotypes. Targeted plasmid integrational mutagenesis was used to disrupt cylB, cylE, cylF and cylH in three wild‐type GBS strains representing serotypes Ia, III and V. Targeted integrations in cylB, cylF and cylH retaining wild‐type haemolytic activity were identified in all strains. In contrast, targeted integrations in cylE were invariably non‐haemolytic and non‐cytolytic, a finding confirmed by in frame allelic exchange of the cylE gene. The haemolytic/cytolytic activity of the cylE allelic exchange mutants could be restored by reintroduction of cylE on a plasmid vector. Inducible expression of cylE, cylF and cylEF demonstrated that it is CylE that confers haemolytic activity in E. coli. We conclude that cylE probably represents the structural gene for the GBS haemolysin/cytolysin, a novel bacterial toxin.
The replicated ftsQAZ and minB chromosomal regions of Escherichia coli segregate on average in line with nucleoid movement
Authors: Roos M. , Van Geel A. B. M. , Aarsman M. E. G. , Veuskens J. T. M. , Woldringh C. L. , Nanninga N.
Published: March 2003

The average cellular positions of the ftsQAZ region (2 min) and the minB region (26.5 min) during the cell cycle was determined by fluorescent in situ hybridization using the position of oriC as a reference point. At the steady‐state growth conditions used, newborn cells had replicated about 50% of the chromosome. By measuring the distances of the labelled oriCs with respect to mid‐cell, we found two well‐separated average oriC positions in cells of newborn length. These average oriC positions moved further apart along with cell elongation. The cellular position of the ftsQAZ gene region resembled the position of oriC, although its average position was closer to mid‐cell. In contrast, a single minB focus was observed at cell birth. Separated minB foci appeared towards the end of DNA replication. The average positions of oriC, ftsQAZ and minB relative to each other fitted a model in which DNA replication takes place in the cell centre and subsequent gene regions pass sequentially through this centre. We have interpreted the polarized orientation of the studied gene regions as a consequence of the mode of DNA segregation.
Genetic and biochemical analyses of a eukaryotic‐like phospholipase D of Pseudomonas aeruginosa suggest horizontal acquisition and a role for persistence in a chronic pulmonary infection model
Authors: Wilderman Paula J. , Vasil Adriana I. , Johnson Zaiga , Vasil Michael L.
Published: March 2003

Phospholipases D (PLDs) are virtually ubiquitous in eukaryotic organisms; however, they are relatively uncommon in prokaryotes. In this report, we demonstrate that the environmentally acquired, opportunistic pathogen Pseudomonas aeruginosa expresses PLD activity. A gene designated pldA was identified in the genomic database of P. aeruginosa PAO1 encoding a protein with significant homology to eukaryotic PLDs, but not to any prokaryotic PLDs. PldA is most homologous to PLDs from mammals and yeast. The pldA gene was cloned and shown to express an ≈ 116 kDa protein with calcium‐regulated PLD activity that is localized to the periplasm. Interestingly, not all strains of P. aeruginosa carry pldA. When present, pldA is always linked to an open reading frame (ORF), ORF4, and a gene (vgrA1) encoding a protein homologous to Vgr from Escherichia coli. Vgr proteins contain regularly repeated dipeptide motifs (valine‐glycine repeats). In E. coli, genes encoding Vgr are associated with multicopy genetic elements designated Rhs (rearrangement hot‐spots). P. aeruginosa PAO1 has 10 vgr homologues dispersed throughout its genome, but the copy number of these genetic elements varies considerably in different strains. Neither vgrA1 nor ORF4 is present in strains lacking pldA. Furthermore, sequences flanking vgrA1, pldA and ORF4 in the P. aeruginosa strains examined are highly conserved, suggesting a specific site of insertion. These and other data suggest that vgrA1, pldA and ORF4 constitute an ≈ 7 kb mobile genetic element and that pldA was acquired horizontally, perhaps from a eukaryotic organism. Competition studies between a PldA knock‐out mutant and the parental wild‐type strain indicate that PldA contributes to the ability of P. aeruginosa PAO1 to persist in a chronic pulmonary infection model in rats.
Two different pathways are involved in the β‐oxidation of n‐alkanoic and n‐phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications
Authors: Olivera Elías R. , Carnicero David , García Belén , Miñambres Baltasar , Moreno Miguel A. , Cañedo Librada , DiRusso Concetta C. , Naharro Germán , Luengo José M.
Published: March 2003

In Pseudomonas putida U, the degradation of n‐alkanoic and n‐phenylalkanoic acids is carried out by two sets of β‐oxidation enzymes (βI and βII). Whereas the first one (called βI) is constitutive and catalyses the degradation of n‐alkanoic and n‐phenylalkanoic acids very efficiently, the other one (βII), which is only expressed when some of the genes encoding βI enzymes are mutated, catabolizes n‐phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the βI genes handicaps the growth of P. putida U in media containing n‐alkanoic or n‐phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n‐alkanoates as a result of the induction of βII, but they were still unable to catabolize n‐phenylalkanoates completely, as the βI‐FadBA enzymes are essential for the β‐oxidation of certain n‐phenylalkanoyl‐CoA derivatives when they reach a critical size. Owing to the existence of the βII system, mutants lacking βIfadB/A are able to synthesize new poly 3‐OH‐n‐alkanoates (PHAs) and poly 3‐OH‐n‐phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.
Non‐growing Escherichia coli cells starved for glucose or phosphate use different mechanisms to survive oxidative stress‡
Authors: Moreau Patrice L. , Gérard Fabien , Lutz Norbert W. , Cozzone Patrick
Published: March 2003

Recent data suggest that superoxide dismutases are important in preventing lethal oxidative damage of proteins in Escherichia coli cells incubated under aerobic, carbon starvation conditions. Here, we show that the alkylhydroperoxide reductase AhpCF (AHP) is specifically required to protect cells incubated under aerobic, phosphate (Pi) starvation conditions. Additional loss of the HP‐I (KatG) hydroperoxidase activity dramatically accelerated the death rate of AHP‐deficient cells. Investigation of the composition of spent culture media indicates that ΔahpCF katG cells leak nutrients, which suggests that membrane lipids are the principal target of peroxides produced in Pi‐starved cells. In fact, the introduction of various mutations inactivating repair activities revealed no obvious role for protein or DNA lesions in the viability of ahp cells. Because the death of ahp cells was directly related to ongoing aerobic glucose metabolism, we wondered how glycolysis, which requires free Pi, could proceed. 31P nuclear magnetic resonance spectra showed that Pi‐starved cells consumed Pi but were apparently able to liberate Pi from phosphorylated products, notably through the synthesis of UDP‐glucose. Whereas expression of the ahpCF and katG genes is enhanced in an OxyR‐dependent manner in response to H2O2 challenge, we found that the inactivation of oxyR and both oxyR and rpoS genes had little effect on the viability of Pi‐starved cells. In stark contrast, the inactivation of both oxyR and rpoS genes dramatically decreased the viability of glucose‐starved cells.
The essential role of the promoter‐proximal subunit of CAP in pap phase variation: Lrp‐ and helical phase‐dependent activation of papBA transcription by CAP from −215
Authors: Weyand Nathan J. , Braaten Bruce A. , Van Der Woude Marjan , Tucker Julie , Low David A.
Published: March 2003

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP–CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP‐dependent activation of papBA requires binding of the leucine‐responsive regulatory protein (Lrp) at binding sites that extend from −180 to −149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP‐dependent promoters. Also, like class I promoters, the C‐terminal domain of the α‐subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an ‘oriented heterodimer’ approach with wild‐type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter‐distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H‐NS. The results presented here show that AR1 of the promoter‐proximal CAP subunit was required for papBA transcription even in the absence of the histone‐like protein H‐NS. These results show that the promoter‐proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C‐terminal domain of the α‐subunit of RNA polymerase.
Mammalian 14‐3‐3β associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG
Authors: Scidmore M. A. , Hackstadt T.
Published: March 2003

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD–G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two‐hybrid screen was employed to identify interacting host proteins. One protein, 14‐3‐3β, was identified that interacted specifically with IncG. The interaction between 14‐3‐3β and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP‐14‐3‐3β fusion protein. 14‐3‐3 proteins are phosphoserine‐binding proteins. Immunoprecipitation studies with [32P]‐orthophosphate‐labelled cells demonstrated that IncG is phosphorylated in both chlamydia‐infected HeLa cells and in yeast cells expressing IncG. Site‐directed mutagenesis of predicted 14‐3‐3 phosphorylation sites demonstrated that IncG binds to 14‐3‐3β via a conserved 14‐3‐3‐binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14‐3‐3β interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14‐3‐3β is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.
No syringes please, ejection of phage T7 DNA from the virion is enzyme driven
Authors: Molineux Ian J.
Published: March 2003

Development of a sensitive assay that measures the rate of cellular internalization of an infecting bacteriophage T7 genome has led to surprising observations on the initiation of infection. Proteins ejected from the phage virion probably function as an extensible tail to form a channel across the cell envelope. This channel is subsequently used for translocating the phage genome into the cell. One of these ejected proteins also controls the amount of DNA that enters the cell, rendering subsequent internalization of the remainder of the genome dependent on transcription. Mutations affecting this protein allow the entire phage genome to enter a cell by the transcription‐independent process. This process exhibits pseudo‐zero‐order reaction kinetics and a temperature dependence of translocation rate that are not expected if DNA ejection from a phage capsid were caused by a physical process. The temperature dependence of transcription‐independent T7 DNA translocation rate is similar to those of enzyme‐catalysed reactions. Current data suggest a highly speculative model, in which two of the proteins ejected from the phage head establish a molecular motor that ratchets the phage genome into the cell.
A set of ftsZ mutants blocked at different stages of cell division in Caulobacter
Authors: Wang Yan , Jones Benjamin D. , Brun Yves V.
Published: March 2003

FtsZ is required throughout the cell division process in eubacteria and in archaea. We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus. Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding. Molecular modelling indicated that all the clustered‐charged‐to‐alanine mutations had altered amino acids at the surface of the protein. Of 13 such mutants, four were recessive‐lethal, three were dominant‐lethal, and six had no discernible phenotype. An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive‐lethal mutations and used to show that they blocked cell division at distinct stages. One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division. The effect of the recessive mutations on the subcellular localization of FtsZ was determined. Models to explain the various mutant phenotypes are discussed. This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.
flp‐1, the first representative of a new pilin gene subfamily, is required for non‐specific adherence of Actinobacillus actinomycetemcomitans
Authors: Kachlany Scott C. , Planet Paul J. , DeSalle Rob , Fine Daniel H. , Figurski David H. , Kaplan Jeffrey B.
Published: March 2003

Actinobacillus actinomycetemcomitans, a Gram‐negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non‐specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small (≈ 6.5 kDa), fibril‐associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High‐resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5‐ to 7‐nm‐diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7‐TAG epitope fused to its C‐terminus was expressed in the wild‐type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7‐TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903φkan to isolate insertion mutations in the flp‐1 gene (formerly designated flp). Mutants with insertions early in flp‐1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp‐1 in trans, thus providing the first evidence that flp‐1 is required for fibril formation and tight, non‐specific adherence. One mutant was found to have an insertion near the 3′ end of flp‐1 that results in the expression of a truncated and altered C‐terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild‐type bacteria. These findings and related observations with the Flp1‐T7‐TAG protein indicate that the C‐terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp‐1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp‐like genes, including the open reading frame (flp‐2, previously designated orfA) immediately downstream of flp‐1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.
Protein regions important for plasminogen activation and inactivation of α2‐antiplasmin in the surface protease Pla of Yersinia pestis
Authors: Kukkonen Maini , Lähteenmäki Kaarina , Suomalainen Marjo , Kalkkinen Nisse , Emödy Levente , Lång Hannu , Korhonen Timo K.
Published: March 2003

The plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla‐expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, α2‐antiplasmin, and the bacteria were found to inactivate α2‐antiplasmin. In contrast, only poor plasminogen activation and no cleavage of α2‐antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A β‐barrel topology model for Pla and OmpT predicted 10 transmembrane β‐strands and five surface‐exposed loops L1–L5. Hybrid Pla–OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and α2‐antiplasmin. Substitution analysis at 25 surface‐located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla‐expressing recombinant E. coli. The mature α‐Pla of 292 amino acids was processed into β‐Pla by an autoprocessing cleavage at residue K262, and residues important for the self‐recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of α2‐antiplasmin. Cleavage of α2‐antiplasmin and plasminogen activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla‐like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of α2‐antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.
Sites of pH regulation of the urea channel of Helicobacter pylori
Authors: Weeks David L. , Sachs George
Published: March 2003

Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability ≈ 300‐fold over that of a non‐polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half‐maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His‐123, His‐131, Asp‐129, Asp‐140, Glu‐138 and Lys‐132 in the second periplasmic loop (PL2) and His‐193 in the C‐terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His‐193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild‐type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.
SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor
Authors: Słomińska Monika , Węgrzyn Alicja , Konopa Grażyna , Skarstad Kirsten , Węgrzyn Grzegorz
Published: March 2003

The SeqA protein is a negative regulator of initiation of DNA replication in the Escherichia coli chromosome. Here, we demonstrate that SeqA stimulates transcription from the bacteriophage λpR promoter both in vivo and in vitro. The activity of the λpL promoter was found not to be affected by this protein. SeqA‐mediated stimulation of pR was dependent on the state of template methylation: transcription was activated on fully methylated and hemimethylated templates but not on an unmethylated template. Using electrophoretic mobility shift assay and electron microscopy, we demonstrated that SeqA interacts specifically with a pR promoter region located on both fully methylated and hemimethylated DNA molecules, but not on unmethylated DNA. The activity of SeqA was found to affect the initiation of λ plasmid replication positively in vivo, probably via pR‐dependent expression of λ replication genes and transcriptional activation of ori λ. We conclude that, apart from its function in the control of DNA replication, SeqA is also a specific transcription factor.
Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of α‐toxin during device‐related infection resolved by direct quantitative transcript analysis
Authors: Goerke Christiane , Fluckiger Ursula , Steinhuber Andrea , Zimmerli Werner , Wolz Christiane
Published: March 2003

The cytotoxic α‐toxin (encoded by hla) of Staphylococcus aureus is regulated by three loci, agr, sarA and sae, in vitro. Here, we assess the regulation of hla in a guinea pig model of device‐related infection by quantifying RNAIII (the effector molecule of agr) and hla directly in exudates accumulating in infected devices without subculturing of the bacteria. LightCycler reverse transcription–polymerase chain reaction (RT–PCR) was used to quantify the transcripts. Strains RN6390 and Newman expressed considerably smaller amounts of RNAIII in the guinea pig than during in vitro growth. The residual RNAIII expression decreased during the course of infection and was negatively correlated with bacterial densities. As with RNAIII, the highest hla expression was detected in both strains early in infection. Even in strain Newman, a weak hla producer in vitro, a pronounced expression of hla was observed during infection. Likewise, four S. aureus isolates from cystic fibrosis (CF) patients expressed hla despite an inactive agr during device‐related infection as in the CF lung. Mutation of agr and sarA in strain Newman and RN6390 had no consequence for hla expression in vivo. In contrast, the mutation in sae resulted in severe downregulation of hla in vitro as well as in vivo. In conclusion, S. aureus seems to be provided with regulatory circuits different from those characterized in vitro to ensure α‐toxin synthesis during infections.
An essential role for phoP in Mycobacterium tuberculosis virulence
Authors: Pérez Esther , Samper Sofía , Bordas Yann , Guilhot Christophe , Gicquel Brigitte , Martín Carlos
Published: March 2003

Two‐component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two‐component systems, such as PhoP/PhoQ, control transcription of key virulence genes essential for survival in host cells in diverse intracellular bacterial pathogens, including Salmonella sp., Shigella sp. and Yersinia sp. In this study, we have disrupted the phoP gene from Mycobacterium tuberculosis, which codes for a putative transcription regulator factor of the two‐component system PhoP/PhoR. The phoP mutant strain exhibited impaired multiplication when cultured in mouse bone marrow‐derived macrophages. However, the mutation did not appear to affect survival of the organisms adversely inside macrophages. The mutant strain was also attenuated in vivo in a mouse infection model, with impaired growth observed in the lungs, livers and spleens. The results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but is not essential for persistence of the bacilli.
A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin
Authors: Dé Emmanuelle , Baslé Arnaud , Jaquinod Michel , Saint Nathalie , Malléa Monique , Molle Gérard , Pagès Jean‐Marie
Published: March 2003

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild‐type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF‐like protein and analysed its pore‐forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild‐type protein and we observed (i) that its single‐channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling β‐lactam diffusion via porins.
TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2
Authors: Rabenau Karen E. , Sohrabi Afshin , Tripathy Ashutosh , Reitter Christopher , Ajioka James W. , Tomley Fiona M. , Carruthers Vern B.
Published: March 2003

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.
Two transporters, TogT and TogMNAB, are responsible for oligogalacturonide uptake in Erwinia chrysanthemi 3937
Authors: Hugouvieux‐Cotte‐Pattat Nicole , Reverchon Sylvie
Published: March 2003

Erwinia chrysanthemi causes soft rot of plants by secreting pectinases which cleave pectin, a polysaccharide cementing the plant cell wall constituents. We demonstrated that two transporters mediate the uptake of the extracellularly formed oligomers in E. chrysanthemi. TogMNAB, a multicomponent transporter member of the ATP‐binding cassette (ABC) superfamily, is only partially responsible for the uptake of pectic oligomers. Its action is completed by that of the second transporter, TogT, a member of the glycoside–pentoside–hexuronide (GPH) family (TC no. 2.2) which includes transporters involved in the uptake of complex sugars, mostly oligosaccharides and glycosides. Each transport system, TogMNAB and TogT, is able to independently mediate the transport of oligogalacturonides and the simultaneous inactivation of both is necessary to give a total absence of growth with pectin as the carbon source. The togT gene constitutes an independent transcriptional unit. Its expression is induced in the presence of pectic derivatives and it is subject to catabolite repression. In vitro, the repressor KdgR and the activator CRP both interact directly with the togT regulatory region. The decreased pathogenicity of single and double togT, togM mutants indicated that a deficiency in uptake of pectic oligomers leads to reduced bacterial multiplication which, in turn, limits plant maceration.
An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid‐encoded regulator, Per
Authors: Shin Sooan , Castanie‐Cornet Marie‐Pierre , Foster John W. , Crawford J. Adam , Brinkley Carl , Kaper James B.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle‐forming pili (BFP) are positively regulated by the plasmid‐encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild‐type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.
Nifs and Sufs in malaria
Authors: Ellis K. E. S. , Clough B. , Saldanha J. W. , Wilson R. J. M. (Iain)
Published: March 2003

This review assembles data from three bodies of literature (bacterial genetics, plastid biogenesis and parasitology) that seldom have much direct cross‐talk. After overcoming terminological complications to sort out microbial nifS from sufS genes, we connect a bacterial operon, recently found to be involved in iron metabolism, the formation of [Fe–S] clusters and oxidative stress to a potentially important gene (sufB) carried on the degenerate plastid genome of malaria and related parasites.
The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence
Authors: Mayorga María E. , Gold Scott E.
Published: March 2003

The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilagobypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein–protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone‐responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone‐responsive MAP kinase cascade and ubc2 is required for pheromone‐responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone‐responsive MAP kinase cascade in U. maydis.
Enterohaemorrhagic and enteropathogenic Escherichia coli use a different Tir‐based mechanism for pedestal formation
Authors: DeVinney Rebekah , Puente Jose Luis , Gauthier Annick , Goosney Danika , Finlay B. Brett
Published: March 2003

Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild‐type EPEC Tir and the non‐tyrosine‐phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.
Molecular characterization of type III secretion signals via analysis of synthetic N‐terminal amino acid sequences
Authors: Lloyd Scott A. , Sjöström Michael , Andersson Sara , Wolf‐Watz Hans
Published: March 2003

Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N‐termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2–8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N‐terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N‐terminal secretion signals. The model accurately classifies the N‐terminal sequences of native type III substrates as efficient secretion signals.
LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion
Authors: Wulff‐Strobel Christine R. , Williams Andrew W. , Straley Susan C.
Published: March 2003

LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ’s effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ’s effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.
Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome
Authors: Josenhans Christine , Niehus Eike , Amersbach Stefanie , Hörster Andrea , Betz Christian , Drescher Bernd , Hughes Kelly T. , Suerbaum Sebastian
Published: March 2003

Helicobacter pylori is thought to regulate gene ex‐pression with a very small set of regulatory genes. We identified a previously unannotated open reading frame (ORF) in the H. pylori 26695 genome (HP1122) as a putative H. pylori flgM gene (σ28 factor antagonist) by a motif‐based bioinformatic approach. Deletion of HP1122 resulted in a fourfold increase in transcription of the σ28‐dependent major flagellin gene flaA, supporting the function of HP1122 as H. pylori FlgM. Helicobacter pylori FlgM lacks a conserved 20‐amino‐acid N‐terminal domain of enterobacterial FlgM proteins, but was able to interact with the Salmonella typhimuriumσ28 (FliA) and inhibit the expression of FliA‐dependent genes in Salmonella. Helicobacter pylori FlgM inhibited FliA to the same extent in a Salmonella strain with an intact flagellar export system and in an export‐deficient strain. Helicobacter pylori FliA was able to drive transcription of FliA‐dependent genes in Salmonella. The effects of mutations in the H. pylori flgM and fliA genes on the H. pylori transcriptome were analysed using whole genome DNA microarrays. The antagonistic roles of FlgM and FliA in controlling the transcription of the major flagellin gene flaA were confirmed, and two additional FliA/FlgM dependent operons (HP472 and HP1051/HP1052) were identified. None of the three genes contained in these operons has a known function in flagellar biogenesis in other bacteria. Like other motile bacteria, H. pylori has a FliA/FlgM pair of sigma and anti‐sigma factors, but the genes controlled by these differ markedly from the Salmonella/Escherichia coli paradigm.
A tyrosine‐phosphorylated 12‐amino‐acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals
Authors: Campellone Kenneth G. , Giese Ndrew , Tipper Onald J. , Leong John M.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12‐residue sequence of EPEC Tir containing Y474 that confers actin‐signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation‐dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.