bluuebunny/crossref_metadata_2025 huggingface dataset:
A Ste6p/P‐glycoprotein homologue from the asexual yeast Candida albicans transports the a‐factor mating pheromone in Saccharomyces cerevisiae
Authors: Raymond Martine , Dignard Daniel , Alarco Anne‐Marie , Mainville Norman , Magee Beatrice B. , Thomas David Y.
Published: March 2003

In Saccharomyces cerevisiae MATa cells, export of the a‐factor mating pheromone is mediated by Ste6p, a member of the ATP‐binding cassette (ABC) superfamily of transporters and a close homologue of mammalian multidrug transporter P‐glycoproteins (Pgps). We have used functional complementation of a ste6Δ mutation to isolate a gene encoding an ABC transporter capable of a‐factor export from the pathogenic yeast, Candida albicans. This gene codes for a 1323‐amino acid protein with an intramolecular duplicated structure, each repeated half containing six potential hydrophobic transmembrane segments and a hydrophilic domain with consensus sequences for an ATP‐binding fold. The predicted protein displays significant sequence similarity to S. cerevisiae Ste6p and mammalian Pgps. The gene has been named HST6, for homologue of STE6. A high degree of structural conservation between the STE6 and the HST6 loci with respect to DNA sequence, physical linkage and transcriptional arrangement indicates that HST6 is the C. albicans orthologue of the S. cerevisiae STE6 gene. We show that the HST6 gene is transcribed in a haploid‐specific manner in S. cerevisiae, consistent with the presence in its promoter of a consensus sequence for Mata1p‐Matα2p binding known to mediate the repression of haploid‐specific genes in S. cerevisiae diploid cells. In C. albicans, HST6 is expressed constitutively at high levels in the different cell types analysed (yeast, hyphae, white and opaque), demonstrating that HST6 transcription is not repressed in this diploid yeast, unlike in diploid S. cerevisiae, and suggesting a basic biological function for the Hst6p transporter in C. albicans. The strong similarity between Hst6p and the multidrug transporter Pgps also raises the possibility that Hst6p could be involved in resistance to antifungal drugs in C. albicans.
The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells
Authors: Braun Laurence , Ohayon Hélène , Cossart Pascale
Published: March 2003

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco‐2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp‐2 and HeLa cells. InlB‐mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3‐kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non‐invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non‐invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes, uptake of InlB‐coated beads required tyrosine phosphorylation in the host cell, PI 3‐kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.
The DevBCA exporter is essential for envelope formation in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Fiedler Gabriele , Arnold Matthias , Hannus Stefan , Maldener Iris
Published: March 2003

The gene devA of the filamentous heterocyst‐forming cyanobacterium Anabaena sp. strain PCC 7120 encodes a protein with high similarity to ATP‐binding cassettes of ABC transporters. Mutant M7 defective in the devA gene is arrested in the development of heterocysts at an early stage and is not able to fix N2 under aerobic conditions. The devA gene is differentially expressed in heterocysts. To gain a better understanding of the structural components of this putative ABC transporter, we determined the complete nucleotide sequence of the entire gene cluster. The two additional genes, named devB and devC, encode proteins with similarities to membrane fusion proteins (DevB) of several ABC exporters and to membrane‐spanning proteins (DevC) of ABC transporters in general. Site‐directed mutations in each of the three genes resulted in identical phenotypes. Heterocyst‐specific glycolipids forming the laminated layer of the envelope were identified in lipid extracts of M7 and in the site‐directed mutants. However, transmission electron microscopy revealed unequivocally that the glycolipid layer is missing in mutant M7. Ultrastructural analysis also confirmed a developmental block at an early stage of differentiation. The results of this study suggest that the devBCA operon encodes an exporter of glycolipids or of an enzyme that is necessary for the formation of the laminated layer. The hypothesis is proposed that an intact envelope could be required for further heterocyst differentiation.
Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors
Authors: De Vries Frits P. , Cole Robert , Dankert Jacob , Frosch Matthias , Van Putten Jos P. M.
Published: March 2003

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified 35SO4‐labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc‐expressing meningococci exploit host cell‐surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28‐producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc‐expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc‐negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.
Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells
Authors: Parida Shreemanta K. , Domann Eugen , Rohde Manfred , Müller Simone , Darji Ayub , Hain Torsten , Wehland Jürgen , Chakraborty Trinad
Published: March 2003

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood–brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild‐type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild‐type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild‐type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non‐invasive mutants dramatically increased their ability to invade HUVECs. In laser‐scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild‐type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.
Aromatic ligand binding and intramolecular signalling of the phenol‐responsive σ54‐dependent regulator DmpR
Authors: O'Neill Eric , Ng Lee Ching , Sze Chun Chau , Shingler Victoria
Published: March 2003

The Pseudomonas‐derived σ54‐dependent regulator DmpR has an amino‐terminal A‐domain controlling the specificity of activation by aromatic effectors, a central C‐domain mediating an ATPase activity essential for transcriptional activation and a carboxy‐terminal D‐domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the −24, −12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A‐domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A‐domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A‐domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 μM. The A‐domain is also shown to have specific affinity for the C‐domain and to repress the C‐domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A‐domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non‐responsiveness of autonomously expressed A‐domain, despite its demonstrated ability to bind phenol, are discussed.
Specific RecA amino acid changes affect RecA–UmuD′C interaction
Authors: Sommer Suzanne , Boudsocq François , Devoret Raymond , Bailone Adriana
Published: March 2003

The UmuD′C mutagenesis complex accumulates slowly and parsimoniously after a 12 J m−2 UV flash to attain after 45 min a low cell concentration between 15 and 60 complexes. Meanwhile, RecA monomers go up to 72 000 monomers. By contrast, when the UmuD′C complex is constitutively produced at a high concentration, it inhibits recombinational repair and then markedly reduces bacterial survival from DNA damage. We have isolated novel recA mutations that enable RecA to resist UmuD′C recombination inhibition. The mutations, named recA [UmuR], are located on the RecA three‐dimensional structure at three sites: (i) the RecA monomer tail domain (four amino acid changes); (ii) the RecA monomer head domain (one amino acid change, which appears to interface with the amino acids in the tail domain); and (iii) in the core of a RecA monomer (one amino acid change). RecA [UmuR] proteins make recombination more efficient in the presence of UmuD′C while SOS mutagenesis is inhibited. The UmuR amino acid changes are located at a head‐tail joint between RecA monomers and some are free to possibly interact with UmuD′C at the tip of a RecA polymer. These two RecA structures may constitute possible sites to which the UmuD′C complex might bind, hampering homologous recombination and favouring SOS mutagenesis.
An RpoS (σS) homologue regulates acylhomoserine lactone‐dependent autoinduction in Ralstonia solanacearum
Authors: Flavier Albert B. , Schell Mark A. , Denny Timothy P.
Published: March 2003

Many bacteria sense an appropriate growth condition or a critical population density for gene expression by producing acylhomoserine lactones (acyl‐HSLs) that act as intercellular autoinduction signals. We recently showed that, in Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium, acyl‐HSL production requires solI, which encodes a putative acyl‐HSL synthase, and that its expression is positively regulated by the acyl‐HSL‐responsive SolR transcriptional regulator. This acyl‐HSL‐dependent autoinduction system is noteworthy because (i) it is regulated by a ‘higher level’ autoinducer system (responsive to 3‐hydroxypalmitic acid methyl ester) via PhcA, a LysR‐type transcriptional regulator and (ii) acyl‐HSL production requires two additional unlinked loci. As reported here, cloning and sequencing of one of these other loci revealed that it encodes a homologue of RpoS, an alternative sigma factor (σS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoSRso) was demonstrated to function as a σ factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS‐dependent bolAp1 gene. Mutation of rpoSRso in R. solanacearum reduced survival during starvation and low pH conditions, but did not affect survival during exposure to hydrogen peroxide, high osmolarity or high temperature. This mutant was also altered in its production of several virulence factors and wilted tomato plants several days more slowly than the wild‐type parent. Transcription of solR and solI were decreased in an rpoSRso background (thereby reducing acyl‐HSL production), but neither mutations in solR, solI or phcA nor addition of acyl‐HSLs affected rpoSRso expression. Therefore, in R. solanacearum the acyl‐HSL‐dependent autoinduction system is controlled both by a second autoinduction system and by the RpoSRso sigma factor.
Isolation and characterization of Fap1, a fimbriae‐associated adhesin ofStreptococcus parasanguisFW213
Authors: Wu Hui , Mintz Keith P. , Ladha Mohammed , Fives‐Taylor Paula M.
Published: March 2003

An adhesin ofStreptococcus parasanguisFW213, a primary colonizer of the tooth surface, has been purified from the culture medium by immunoaffinity chromatography. The purified protein has a molecular mass of 200 kDa and stains positively for carbohydrate. The amino‐terminal sequence indicated that this protein represented a unique streptococcal surface protein. Immunogold labelling of the bacterium indicated that this protein was associated with fimbriae and designated Fap1 (fimbriae‐associated protein). A polymerase chain reaction (PCR) product based on the amino terminus of Fap1 was used to probe an FW213 genomic library. A 9 kb fragment containing thefap1gene was isolated and 2.5 kb have been sequenced. Generation offap1mutants by a single cross‐over (Campbell insertion) or a non‐polar allelic exchange abolished the expression of Fap1. The inactivation offap1resulted in a dramatic reduction in the expression of the long peritrichous fimbriae and adhesion to saliva‐coated hydroxylapatite (SHA). Northern blots probed with an internal gene fragment offap1hybridized to a 9 kb transcript, which suggests thatfap1is transcribed as a polycistronic message. These data demonstrate that Fap1 is a unique streptococcal adhesin that is involved in the assembly ofS.parasanguisFW213 fimbriae and adhesion to SHA.
Distinct roles of an alternative sigma factor during both free‐swimming and colonizing phases of the Vibrio cholerae pathogenic cycle
Authors: Klose Karl E. , Mekalanos John J.
Published: March 2003

Vibrio cholerae, the bacterium that causes cholera, has a pathogenic cycle consisting of a free‐swimming phase outside its host, and a sessile virulent phase when colonizing the human small intestine. We have cloned the V. cholerae homologue of the rpoN gene (encoding σ54) and determined its role in the cholera pathogenic cycle by constructing an rpoN null mutant. The V. cholerae rpoN mutant is non‐motile; examination of this mutant by electron microscopy revealed that it lacks a flagellum. In addition to flagellar synthesis, σ54 is involved in glutamine synthetase expression. Moreover, the rpoN mutant is defective for colonization in an infant mouse model of cholera. We present evidence that the colonization defect is distinct from the non‐motile and Gln phenotypes of the rpoN mutant, implicating multiple and distinct roles of σ54 during the V. cholerae pathogenic cycle. RNA polymerase containing σ54 (σ54‐holoenzyme) has an absolute requirement for an activator protein to initiate transcription. We have identified three regulatory genes, flrABC (flagellar regulatory proteins ABC ) that are additionally required for flagellar synthesis. The flrA and flrC gene products are σ54‐activators and form a flagellar transcription cascade. flrA and flrC mutants are also defective for colonization; this phenotype is probably independent of non‐motility. An flrC constitutive mutation (M114→I) was isolated that is independent of its cognate kinase FlrB. Expression of the constitutive FlrCM114→I from the cholera toxin promoter resulted in a change in cell morphology, implicating involvement of FlrC in cell division. Thus, σ54‐holoenzyme, FlrA and FlrC transcribe genes for flagellar synthesis and possibly cell division during the free‐swimming phase of the V. cholerae life cycle, and some as yet unidentified gene(s) that aid colonization within the host.
The ArcA/ArcB two‐component regulatory system of Escherichia coli is essential for Xer site‐specific recombination at psi
Authors: Colloms Sean D. , Alén Claudia , Sherratt David J.
Published: March 2003

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and ColE1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi. Here, we demonstrate that the ArcA/ArcB two‐component regulatory system of Escherichia coli, which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi. Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.
The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG
Authors: Daefler Simon , Russel Marjorie
Published: March 2003

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C‐terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV‐′InvG43 protein becomes dependent on the co‐expression of another gene, invH, for function in phage assembly. [3H]‐palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV‐′InvG43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.
Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein
Authors: Tønjum Tone , Caugant Dominique A. , Dunham Steve A. , Koomey Michael
Published: March 2003

Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PilQ expression in Neisseria meningitidis (Mc) and found that PilQ− mutants were non‐piliated and deficient in the expression of pilus‐associated phenotypes. Sequence analysis of the 5′ portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR‐generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.
PTR3, a novel gene mediating amino acid‐inducible regulation of peptide transport in Saccharomyces cerevisiae
Authors: Barnes David , Lai Wing , Breslav Michael , Naider Fred , Becker Jeffrey M.
Published: March 2003

We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid‐inducible peptide transport. PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database. Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid‐induced levels of peptide transport. PTR3 was required for amino acid‐induced expression of PTR2, the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression. It was determined that PTR3 also modulates expression of BAP2, the gene encoding the branched‐amino acid permease. Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system.
On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi
Authors: Delbac Frédéric , Peyret Pierre , Méténier Guy , David Danielle , Danchin Antoine , Vivarès Christian P.
Published: March 2003

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two‐dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline‐rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26‐amino‐acid repeats. An N‐terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.
Global regulation by the small RNA‐binding protein CsrA and the non‐coding RNA molecule CsrB
Authors: Romeo Tony
Published: March 2003

Csr (carbon storage regulator) is a recently discovered global regulatory system that controls bacterial gene expression post‐transcriptionally. Its effector is a small RNA‐binding protein referred to as CsrA or, in phytopathogenic Erwinia species, RsmA (repressor of stationary phase metabolites). Numerous genes whose expression occurs in the stationary phase of growth are repressed by csrA/rsmA, and csrA activates certain exponential‐phase metabolic pathways. Glycogen synthesis and catabolism, gluconeogenesis, glycolysis, motility, cell surface properties and adherence are modulated by csrA in Escherichia coli, while the production of several secreted virulence factors, the plant hypersensitive response elicitor HrpNEcc and, potentially, other secondary metabolites are regulated by rsmA in Erwinia carotovora. CsrA represses glycogen synthesis by binding to and destabilizing glgCAP mRNA and is hypothesized to repress other genes by a similar mechanism. The second component of the Csr system is CsrB (AepH in Erwinia species), a non‐coding RNA molecule that forms a large globular ribonucleoprotein complex with approximately 18 CsrA subunits and antagonizes the effects of CsrA in vivoHighly repeated sequence elements found within the loops of predicted stem–loops and other single‐stranded segments of CsrB RNA may facilitate CsrA binding. Current information supports a model in which CsrA exists in an equilibrium between CsrB and CsrA‐regulated mRNAs, which predicts that CsrB levels may be a key determinant of CsrA activity in the cell. The presence of csrA homologues in phylogenetically diverse species further suggests that this novel kind of regulatory system is likely to play a broad role in modulating eubacterial gene expression.
The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S‐dependent promoter in the ddlB–ftsA region
Authors: Ballesteros Manuel , Kusano Shuichi , Ishihama Akira , Vicente Miguel
Published: March 2003

The most potent promoters in the ddlB–ftsA region of the dcw cluster have been analysed for σS‐dependent transcription. Only the gearbox promoter ftsQ1p was found to be transcribed in vitro by RNA polymerase holoenzyme coupled to σS (EσS). This dependency on σS was also found in vivo when single‐copy fusions to a reporter gene were analysed in rpoS and rpoS+ backgrounds. Although ftsQ1p can be transcribed by RNA polymerase containing either σD or σS, there is a preference for EσS when the assay conditions include potassium glutamate and supercoiled templates, a property shared with the bolA1p gearbox promoter. The rest of the promoters assayed, ftsQ2p and ftsZ2p3p4p, similarly to the control bolA2p promoter, were preferentially transcribed by EσD, the housekeeper polymerase. The ftsQ1p and the bolA1p promoters also share the presence of AT‐rich sequences upstream of the − 35 region and the requirement for an intact wild‐type α‐subunit for a proficient transcription, allowing their joint classification as gearboxes.
New tools in an old trade: CS1 pilus morphogenesis
Authors: Sakellaris Harry , Scott June R.
Published: March 2003

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram‐negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone‐like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well‐characterized Pap (pyelonephritis‐associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein ‘tools’ in the old ‘trade’ of forming functional pili.
Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae
Authors: De Boer Marco , Bebelman Jan‐Paul , Gonçalves Paula M. , Maat Jan , Van Heerikhuizen Harm , Planta Rudi J.
Published: March 2003

The BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high‐affinity permease for branched‐chain amino acids. In this paper, we show that, like BAP2, the expression of the BAP3 gene in S. cerevisiae is induced by the addition of branched‐chain amino acids to the medium. Unexpectedly, most other naturally occurring L‐amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3. The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre‐tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1 − mutant. The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a Δleu3 mutant. By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from − 418 to − 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p‐dependent induction of BAP3 expression by (most) amino acids. We have therefore named this sequence UASaa (amino acid‐dependent upstream activator sequence). Neither Stp1p nor Leu3p appear to bind to the UASaa, at least in vitro, as judged from gel retardation assays. Sequences similar to the UASaa can be found in the promoters of BAP2, PTR2 and TAT1 ; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.
Intracellular expression of the ADP‐ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells
Authors: Pederson Kristin J. , Barbieri Joseph T.
Published: March 2003

Exoenzyme S of Pseudomonas aeruginosa is an ADP‐ribosyltransferase, which is secreted via a type III‐dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino‐terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS‐dependent ADP‐ribosyltransferase activity to the carboxyl‐terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP‐ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl‐terminal 222 amino acids of exoenzyme S, which represent the FAS‐dependent ADP‐ribosyltransferase domain (termed ΔN222), and a point mutant, ΔN222‐E381A, which possesses a 2000‐fold reduction in the capacity to ADP‐ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with ΔN222 expressed ADP‐ribosyltransferase activity. Co‐transfection of ΔN222, but not ΔN222‐E381A, resulted in a decrease in the steady‐state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with ΔN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either ΔN222‐E381A or control plasmid. Together, these data indicate that expression of the ADP‐ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.
Nitrogen control of the glnN gene that codes for GS type III, the only glutamine synthetase in the cyanobacterium Pseudanabaena sp. PCC 6903
Authors: Crespo José L. , García‐Domínguez Mario , Florencio Francisco J.
Published: March 2003

Pseudanabaena
sp. strain PCC 6903 is the first cyanobacteria lacking the typical prokaryotic glutamine synthetase type I encoded by the
glnA
gene. The
glnN
gene product, glutamine synthetase type III, is the only glutamine synthetase activity present in this cyanobacterium. Analysis of
glnN
expression clearly indicated a nitrogen‐dependent regulation.
Pseudanabaena glnN
gene expression and GSIII activity were upregulated under nitrogen starvation or using nitrate as a nitrogen source, while low levels of transcript and activity were found in ammonium‐containing medium. Primer extension analysis showed that the
glnN
gene promoter structure resembled that of the NtcA‐related promoters. Mobility shift assays demonstrated that
Synechocystis
sp. PCC 6803 NtcA protein, expressed and purified from
Escherichia coli
, bound to the promoter of the
Pseudanabaena
6903
glnN
gene. The NtcA control of the
glnN
gene in this cyanobacterium suggested that, in the absence of a
glnA
gene, NtcA took control of the only glutamine synthetase gene in a fashion similar to the way the
glnA
gene is governed in those cyanobacteria harbouring a
glnA gene.
When drug inactivation renders the target irrelevant to antibiotic resistance: a case story with β‐lactams
Authors: Lakaye Bernard , Dubus Alain , Lepage Sophie , Groslambert S. , Frère Jean‐Marie
Published: March 2003

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a β‐lactamase‐overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor β‐lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.
Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD
Authors: Neyt Cécile , Cornelis Guy R.
Published: March 2003

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST–YopB hybrid proteins and SycD in Escherichia coli, we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST–YopB in E. coli. By analysis of a series of truncated GST–YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia. In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB–YopD and LcrV.
ComEA is a DNA receptor for transformation of competent Bacillus subtilis
Authors: Provvedi R. , Dubnau D.
Published: March 2003

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high‐affinity DNA binding. A domain necessary for DNA binding is located at the C‐terminus of ComEA. Proteins with similar 60–80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double‐stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA‐binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.
Borrelia burgdorferi erpT expression in the arthropod vector and murine host
Authors: Fikrig Erol , Chen Manchuan , Barthold Stephen W. , Anguita Juan , Feng Wen , Telford Sam R. , Flavell Richard A.
Published: March 2003

The expression of a Borrelia burgdorferi gene, erpTwas investigated throughout the spirochaete life cycle in the arthropod vector and the murine host. Three phage clones from a B. burgdorferi DNA expression library synthesized a 30 kDa antigen that was recognized by antibodies in the sera of B. burgdorferi‐infected mice but not mice hyperimmunized with B. burgdorferi lysates. Differential antibody binding suggested that this protein was preferentially expressed in vivo. This antigen was designated ErpT, based upon 99.6% homology with the BBF01 sequence in the B. burgdorferi genome. ErpT was not detected on spirochaetes cultured in BSK II medium by indirect immunofluorescence or in B. burgdorferi lysates by immunoblotting, implying that ErpT is not readily produced in vitro. erpT mRNA was not discernible by Northern blot but was identified by RNA polymerase chain reaction in vitro, indicating that erpT is expressed at low levels by cultured spirochaetes. erpT expression was then investigated in the vector and mice because B. burgdorferi do not normally reside in culture medium. RNA polymerase chain reaction and immunofluorescence studies demonstrated that erpT was expressed by a small minority of B. burgdorferi (11/500, 2.2%) within unfed ticks and then repressed during engorgement. erpT mRNA or ErpT antibodies were first detected in B. burgdorferi‐infected mice at 4 weeks, suggesting that erpT was not expressed in the early stages of murine infection. Then, during persistent infection, RNA polymerase chain reaction showed that erpT was expressed by B. burgdorferi within the joints, heart and spleen, but not by spirochaetes in the skin. Immunization of mice with ErpT was antigenic but was not protective. These studies demonstrate that B. burgdorferi erpT is differentially expressed throughout the B. burgdorferi life cycle, in both the vector and the mammalian host, and is primarily expressed in extracutaneous sites during murine infection.
Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli
Authors: Lewis Dale E. A. , Geanacopoulos Mark , Adhya Sankar
Published: March 2003

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping‐mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone‐like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA–multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone‐like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.
Insertion of Escherichia coliα‐haemolysin in lipid bilayers as a non‐transmembrane integral protein: prediction and experiment
Authors: Soloaga Ana , Veiga M. Pilar , García‐Segura Luis M. , Ostolaza Helena , Brasseur Robert , Goñi Félix M.
Published: March 2003

α‐Haemolysin is an extracellular protein toxin (≈107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor‐mediated binding is not essential. In this work, we have studied the perturbation produced by purified α‐haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X‐114, and freeze–fracture electron microscopy. All the data concur in indicating that α‐haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted α‐haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic α‐helices, appropriate for protein–lipid interaction, but no hydrophobic transmembrane helices are predicted in α‐haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by α‐haemolysin; in particular, a non‐transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of α‐haemolysin as a pore‐forming toxin.
Chemotactic‐like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY
Authors: Barak Rina , Eisenbach Michael
Published: March 2003

We describe a chemotactic‐like response of Escherichia coli strains lacking most of the known chemotaxis machinery but containing high levels of the response regulator CheY. The bacteria accumulated in aspartate‐containing capillaries, they formed rings on tryptone‐containing semisolid agar, and the probability of counterclockwise flagellar rotation transiently increased in response to stimulation with aspartate (10−10–10−5 M; the response was inverted at > 10−4 M). The temporal response was partial and delayed, as was the response of a control wild‐type strain having a high CheY level. α‐Methyl‐DL‐aspartate, a non‐metabolizable analogue of aspartate as well as other known attractants of E. Coli, glucose and, to a lesser extent, galactose, maltose and serine caused a similar response. So did low concentrations of acetate and benzoate (which, at higher concentrations, act as repellents for wild‐type E. coli ). Other tested repellents such as indole, Ni2+ and Co2+ increased the clockwise bias. These observations raise the possibility that, at least when the conventional signal transduction components are missing, a non‐conventional chemotactic signal transduction pathway might be functional in E. coli. Potential molecular mechanisms are discussed.
Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30)
Authors: Drummelsmith Jolyne , Whitfield Chris
Published: March 2003

The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high‐molecular‐weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A‐core in a lipopolysaccharide molecule (K30LPS). To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized. This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen. The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST(1) exporter (Wzx), required to transfer lipid‐linked K30 units across the plasma membrane to the periplasmic space; and a K30‐antigen polymerase (Wzy). These gene products are typical of those seen in O‐antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface. The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule. Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides. Mutant analysis indicates that Wza and Wzc are required for wild‐type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS. These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.