bluuebunny/crossref_metadata_2025 huggingface dataset:
Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion
Authors: Abe Akio , De Grado Myriam , Pfuetzner Richard A. , Sánchez‐SanMartín Claudia , DeVinney Rebekah , Puente José Luis , Strynadka Natalie C. J. , Finlay B. Brett
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli‐secreted proteins) that are required for full virulence. Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal‐like structure. In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post‐transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino‐terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir–CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti‐degradation factor by specifically binding to its amino‐terminus, forming a multimeric stabilized complex.
Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide‐inducible promoters
Authors: Wood Timothy I. , Griffith Kevin L. , Fawcett William P. , Jair Kam‐Wing , Schneider Thomas D. , Wolf Richard E.
Published: March 2003

SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS‐dependent promoters, e.g. zwf and fpr, whose SoxS binding sites (‘soxbox’) lie upstream of the −35 region of the promoter, activation requires the C‐terminal domain of the RNA polymerase α‐subunit, while at class II SoxS‐dependent promoters, e.g. fumC and micF, whose binding sites overlap the −35 region, activation is independent of the α‐CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase‐dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf−35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr−35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.
Antisense inhibition of amoebapore expression inEntamoeba histolyticacauses a decrease in amoebic virulence
Authors: Bracha Rivka , Nuchamowitz Yael , Leippe Matthias , Mirelman David
Published: March 2003

Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasiteEntamoeba histolytica, which is responsible for the killing of target cells. These 77‐residue peptides are structural and functional analogues of NK‐lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP‐R2) containing theNeoresistance gene and the gene coding for amoebapore A, including its 5′ and 3′ untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of theE. histolyticaribosomal protein (RP‐L21) gene copies. Transfectants of virulentE. histolyticastrain HM‐1:IMSS, in which the expression of amoebapore was inhibited by ~ 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP‐R2 transfectant displayed lower pore‐forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP‐R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites ofE. histolyticaexert their remarkable cytolytic and tissue destructive activity.
Testing the ‘+2 rule’ for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection
Authors: Seydel Anke , Gounon Pierre , Pugsley Anthony P.
Published: March 2003

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose‐binding protein (MalE), cannot use maltose unless a wild‐type copy of malE is present in trans. Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane‐anchored lipoprotein resulted in N‐terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose‐using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid‐borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K‐12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane.
The cell wall architecture of Candida albicans wild‐type cells and cell wall‐defective mutants
Authors: Kapteyn J. C. , Hoyer L. L. , Hecht J. E. , Müller W. H. , Andel A. , Verkleij A. J. , Makarow M. , Van Den Ende H. , Klis F. M.
Published: March 2003

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.
Phase variation of Ag43 in Escherichia coli: Dam‐dependent methylation abrogates OxyR binding and OxyR‐mediated repression of transcription
Authors: Haagmans W. , Van Der Woude M.
Published: March 2003

It has been shown previously that phase variation of the outer membrane protein Antigen43 (Ag43) of Escherichia coli requires the DNA‐methylating enzyme deoxyadenosine methyltransferase (Dam) and the global regulator OxyR. In this study, we analysed the regulation of the Ag43 encoding gene (agn) using isolates containing a fusion of the agn regulatory region to the reporter gene lacZ. Our results indicate that phase variation of Ag43 is regulated at the level of transcription. Repression of agn′–lacZ transcription required OxyR, whereas activation required Dam. The regulatory region of agn contains three GATC sequences, which are target sites for Dam‐dependent methylation. In vivo, the methylation state of these GATC sequences correlated with the transcription state of agn′–lacZ. These GATC sequences were not protected from Dam‐dependent methylation in an oxyR background, suggesting that OxyR binding results in Dam‐dependent methylation protection in OFF cells. In vitro, both oxidized OxyR and OxyR(C199S), which is locked in the reduced conformation, bound to the agn regulatory region, but methylation of the three GATC sequences abrogated this binding. In vivo, OxyR(C199S) was sufficient to repress Ag43 transcription. Our data support a model in which OxyR‐mediated repression of transcription is alleviated by methylation of three GATC sequences in its binding site. In addition, we show that, in an oxyR background, Dam was still required for full activation, suggesting that the model concerning the role of Dam in agn regulation is incomplete. These results show that Dam‐dependent phase variation in E. coli is not limited to the previously identified regulatory system of the family of pap‐like fimbrial operons.
The essential protein Fap7 is involved in the oxidative stress response of Saccharomyces cerevisiae
Authors: Juhnke Holger , Charizanis Christina , Latifi Faiaz , Krems Bernhard , Entian Karl‐Dieter
Published: March 2003

Pos9 (Skn7) is an important transcription factor that, together with Yap1, induces the expression of oxidative stress target genes in Saccharomyces cerevisiae. The activation of Pos9 upon an oxidative stress signal occurs post‐translationally. In a mutant screen for factors involved in the activation of a Pos9‐dependent reporter gene upon oxidative stress, we identified the mutant fap7‐1 (for factor activating Pos9). This point mutant failed to activate a Gal4‐Pos9 hybrid transcription factor, assayed by hydrogen peroxide‐induced GAL1‐lacZ reporter gene activities. Additionally, the fap7‐1 mutant strain was sensitive to oxidative stress and revealed slow growth on glucose compared with the wild type. The fap7‐1 mutation also affected the induction of the Pos9 target gene TPX1 and of a synthetic promoter previously identified to be regulated in a Yap1‐ and Pos9‐dependent manner. This lack of induction was specific as the fap7‐1 mutant response to other stresses such as sodium chloride or co‐application of both hydrogen peroxide and sodium chloride was not affected, as tested with the Pos9‐independent expression pattern of a TPS2‐lacZ reporter system. We identified the gene YDL166c to be allelic to the FAP7 gene and to be essential. Fluorescence microscopy of Fap7–GFP fusion proteins indicated a nuclear localization of the Fap7 protein. Our data suggest that Fap7 is a nuclear factor important for Pos9‐dependent target gene transcription upon oxidative stress.
The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes
Authors: Gaillot Olivier , Pellegrini Elisabeth , Bregenholt Søren , Nair Shamila , Berche Patrick
Published: March 2003

We identified the stress‐induced ClpP of Listeria monocytogenes and demonstrated its crucial role in intracellular survival of this pathogen. ClpP is a 21.6 kDa protein belonging to a family of proteases highly conserved in prokaryotes and eukaryotes. A clpP‐deleted mutant enabled us to demonstrate that ClpP is involved in proteolysis and is required for growth under stress conditions. Intramacrophage survival of this mutant was strongly restricted, thus resulting in loss of virulence for the mouse. The activity of listeriolysin O, a major virulence factor implicated in bacterial escape from phagosomes of macrophages, was much reduced in the clpP mutant under stress conditions. Direct evidence for the role of ClpP in the intracellular parasitism was obtained by showing that virulence and haemolytic activity were fully restored by complementation of the mutant. These results suggest that ClpP is involved in the rapid adaptive response of intracellular pathogens during the infectious process.
The bacteriophage T4 anti‐sigma factor AsiA is not necessary for the inhibition of early promoters in vivo
Authors: Pène Carole , Uzan Marc
Published: March 2003

Bacteriophage T4 early promoters are utilized immediately after infection and are abruptly turned off 2–3 min later (at 30°C) when the middle promoters are activated. The viral early protein AsiA has been suspected to bring about this transcriptional switch: not only does it activate transcription at middle promoters in vivo and in vitro but it also shows potent anti‐σ70 activity in vitro, suggesting that it is responsible for the shut‐off of early transcription. We show here that after infection with a phage deleted for the asiA gene the inhibition of early transcription occurs to the same extent and with the same kinetics as in a wild‐type infection. Thus, another AsiA‐independent circuit efficiently turns off early transcription. The association of a mutation in asiA with a mutation in mod, rpbA, motA or motB has no effect on the inhibition of early promoters, showing that none of these phage‐encoded transcriptional regulators is necessary for AsiA‐independent shut‐off. It is not known whether AsiA is able to inhibit early promoters in vivo, but host transcription is strongly inhibited in vivo upon induction of AsiA from a multicopy plasmid.
A member of the Plasmodium falciparum Pf60 multigene family codes for a nuclear protein expressed by readthrough of an internal stop codon
Authors: Bischoff E. , Guillotte M. , Mercereau‐Puijalon O. , Bonnefoy S.
Published: March 2003

Four large multigene families have been described in Plasmodium falciparum malaria parasites (var, rif, stevor and Pf60). var and rif genes code for erythrocyte surface proteins and undergo clonal antigenic variation. We report here the characterization of the first Pf60 gene. The 6.1 gene is constitutively expressed by all mature blood stages and codes for a protein located within the nucleus. It has a single copy, 7‐exon, 5′ domain, separated by an internal stop codon from a 3′ domain that presents a high homology with var exon II. Double‐site immunoassay and P. falciparum transient transfection using the reporter luciferase gene demonstrated translation through the internal ochre codon. The 6.1 N‐terminal domain has no homology with any protein described to date. Sequence analysis identified a leucine zipper and a putative nuclear localization signal and showed a high probability for coiled coils. Evidence for N‐terminal coiled coil‐mediated protein interactions was obtained. This identifies the 6.1 protein as a novel nuclear protein. These data show that the Pf60 and var genes form a superfamily with a common 3′ domain, possibly involved in regulating homo‐ or heteromeric interactions.
In vivo studies of upstream regulatory cis‐acting elements of the alcR gene encoding the transactivator of the ethanol regulon in Aspergillus nidulans
Authors: Mathieu Martine , Fillinger Sabine , Felenbok B.
Published: March 2003

The alcR gene of Aspergillus nidulans, which encodes the specific transactivator of the ethanol utilization pathway, is positively autoregulated and carbon catabolite repressed. Regulation by these two circuits occurs at the transcriptional level via the binding of the two regulators, AlcR and CreA, to their cognate targets respectively. We demonstrate here that out of two clustered putative AlcR repeated consensus sequences, only the palindromic target is functional in vivo. Hence, it is solely responsible for the alcR positive autogenous activation loop. Transcript mapping of the alcR gene showed that transcription initiation can occur at 553 bp and at or near 86 bp upstream of the start codon. These transcription start sites yield a transcript of 3.0 kb, which appears only under induced growth conditions, and of 2.6 kb, which is present under both induced and non‐induced growth conditions respectively. Nine CreA consensus sites are present in the alcR promoter but only two pairs of two sites are functional in vivo. One of them is located in close proximity to the AlcR functional target. Within this pair, both sites are necessary to mediate a partial repression of alcR transcription. Disruption of either site results in an overexpression of alcR due to the absence of direct competition between AlcR and CreA for the same DNA region. The second functional pair of CreA sites is located between the two transcription initiation sites. Disruption of either of the two sites results in a totally derepressed alcR transcription, showing that they work as a pair constituting the more efficient repression mechanism. Thus, CreA acts by two different mechanisms: by competing with AlcR for the same DNA region and by an efficient direct repression. The latter mechanism presumably interfers with the general transcriptional machinery.
Phase variation of the gonococcal siderophore receptor FetA
Authors: Carson Susan D. Biegel , Stone Barbara , Beucher Margaret , Fu Jennifer , Sparling P. Frederick
Published: March 2003

FetA, the recently characterized gonococcal ferric enterobactin receptor, exhibited extremely rapid phase variation between high‐ and low‐expression levels. The frequency of phase variation was ≈ 1.3% in both directions in gonococcal strain FA1090. FetA expression in the ‘high phase’ was significantly greater than the level of expression in the ‘low phase’. Expression levels correlated with the number of cytosine residues in a string of cytosines located close to the transcriptional start site for fetA between the putative −10 and −35 consensus sequences. Antibody production against FetA commonly occurs in infected patients, and we therefore hypothesize that phase variation reflects a balance between the advantages of being able to use a ferric siderophore as an iron source and evasion of the host immune response.
AglU, a protein required for gliding motility and spore maturation of Myxococcus xanthus, is related to WD‐repeat proteins
Authors: White David J. , Hartzell Patricia L.
Published: March 2003

The aglU gene of Myxococcus xanthus encodes a protein similar to Het‐E1 (vegetative incompatibility) from Podospora anserina, acylaminoacyl‐peptidase from Bacillus subtilis, and TolB from Escherichia coli. These proteins all have evenly spaced SPDG repeats that are characteristic of a larger motif called the WD‐repeat. The WD‐repeat is predicted to form a β‐propeller structure that mediates the assembly of heteromeric protein complexes. AglU has a consensus lipoprotein attachment motif that includes a type II signal sequence followed by a cysteine residue. This suggests that AglU is matured, then attached to the outer membrane via fatty acid acylation at this Cys. Cells carrying a mutation in aglU are blocked in adventurous gliding and can swarm only if cells are in contact with one another. When starved of nutrients, the aglU mutant aggregates and forms multicellular fruiting bodies like the wild‐type strain, but is unable to produce heat‐resistant spores. This suggests that adventurous gliding motility, per se, is not required for development, but that AglU is essential for a terminal step of spore differentiation.
Identification of the urease operon in Helicobacter pylori and its control by mRNA decay in response to pH
Authors: Akada Junko K. , Shirai Mutsunori , Takeuchi Hiroaki , Tsuda Masataka , Nakazawa Teruko
Published: March 2003

We investigated the transcription of the urease gene cluster ureABIEFGH in Helicobacter pylori to determine the regulation of gene expression of the highly produced enzyme urease. Northern blot hybridization analysis demonstrated that cells of the wild‐type strain grown in an ordinary broth had transcripts of ureAB, ureABI, ureI, ureIE′ and ure′FGH, but cells of a ureI‐disrupted mutant had only the ureAB transcript. When the wild‐type cells were exposed to pH 8 for 30 min, very little mRNA was detected. However, when exposed to pH 6, a large amount of the ureIE′′ transcript, which was longer than the ureIE′ transcript, together with the additional transcripts ureABIEFGH and ure′EFGH were detected. Rifampicin addition experiments demonstrated that urease mRNAs, and the ureIE′ transcripts in particular, are more stable at pH 5.5 than at pH 7. In accord with these results, urease activity in the crude cell extract of the pH 5.5 culture was twice as much as that of the pH 7 culture, although the amounts of UreA and UreB detected by immunoblot analysis were similar. The transcription start point of ureI was identified by primer extension using a ureA promoter‐deleted mutant, and a consensus sequence of RpoD‐RNA polymerase was found in the ureI promoter. The 3′ end of the ureIE′′ mRNA, determined using S1 nuclease mapping, revealed that the transcript is able to cover the majority of the ureE open reading frame (ORF) that might be sufficient for UreE activity. Based on the above results, we conclude that the urease gene cluster of H. pylori consists of two operons, ureAB and ureIEFGH, and that primary transcripts of the latter as well as the read‐through transcript, ureABIEFGH, are cleaved to produce several species of mRNA. It has been suggested that the ureIEFGH operon is regulated post‐transcriptionally by mRNA decay in response to environmental pH. We are tempted to speculate that the ureE′′ transcript present in acidic pH may contribute to produce an active product that can proceed the nickel incorporation to the active centre, the final step of urease biosynthesis.
Identification of a target gene for the bE–bW homeodomain protein complex inUstilago maydis
Authors: Romeis Tina , Brachmann Andreas , Kahmann Regine , Kämper Jörg
Published: March 2003

In the phytopathogenic fungusUstilago maydis, sexual and pathogenic development are controlled by the multiallelicbmating type locus. Theblocus encodes a pair of unrelated homeodomain proteins termed bE and bW that form a heterodimeric complex when both proteins originate from different alleles. The heterodimer is presumed to be the central regulator for pathogenicity genes. Here, we show that a translational fusion protein comprising specific domains of bE1 and bW2 remains biologically active and binds to a sequence motif in the promoter oflga2, a gene located in theamating type locus. Thisbbindingsequence1(bbs1) is also recognized by the native bE1–bW2 heterodimerin vivoand mediates theb‐dependent regulation of thelga2gene.Our data demonstrate that the bE–bW heterodimer can act as a positive transcriptional regulator.
Evidence for a second function forSaccharomyces cerevisiaeRev1p
Authors: Nelson John R. , Gibbs Peter E. M. , Nowicka Adrianna M. , Hinkle David C. , Lawrence Christopher W.
Published: March 2003

The function of theSaccharomyces cerevisiae REV1gene is required for translesion replication and mutagenesis induced by a wide variety of DNA‐damaging agents. We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic sitein vivo. However, we now find that Rev1p function is needed for the bypass of a T–T (6–4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function. In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site. However, replication past this lesion was also much reduced in the G‐193Rrev1‐1mutant, which we find retains substantial levels of deoxycytidyl transferase activity. This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function. The bypass of an abasic site and T–T (6–4) lesion also depended onREV3function, but neither it norREV1was required for replication past the T–T dimer; bypass of this lesion presumably depends on another enzyme.
Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery
Authors: Justice Sheryl S. , García‐Lara Jorge , Rothfield Lawrence I.
Published: March 2003

SulA and MinCD are specific inhibitors of cell division in Escherichia coli. In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented the formation of high‐molecular‐weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo. However, MinCD did prevent the formation of FtsA rings. Increased expression of ftsA suppressed MinCD‐induced division inhibition, but had no effect on SulA‐induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.
Mutations conferring amino acid residue substitutions in the carboxy‐terminal domain of RNA polymerase α can suppress clpX and clpP with respect to developmentally regulated transcription in Bacillus subtilis
Authors: Nakano Michiko M. , Zhu Yi , Liu Jiajian , Reyes Dindo Y. , Yoshikawa Hirofumi , Zuber Peter
Published: March 2003

The Bacillus subtilis clpX and clpP genes are the sites of pleiotropic mutations that adversely affect growth on a variety of media and impair developmental processes such as sporulation and competence development. ClpX is necessary for the post‐exponential induction of genes that require the σH form of RNA polymerase for transcription. Both ClpX and ClpP are required for the activation of σA‐dependent transcription of the srf operon that encodes surfactin synthetase and the regulatory peptide ComS, required for the development of genetic competence. Transcription of srf is activated by the two‐component regulatory system ComPA in response to the peptide pheromone, ComX, which mediates cell density‐dependent control. A clpX mutant, although able to produce ComX, is unable to respond to the pheromone. A mutant allele of comP, encoding a product whose activity is independent of ComX, is not able to suppress clpX with respect to srf expression, suggesting that ClpXP acts at the level of ComA‐dependent activation of srf transcription initiation. Suppressor mutations of clpX (cxs‐1 and cxs‐2) were isolated in screens for pseudorevertants exhibiting high levels of srf expression and σH‐dependent transcription respectively. One mutation, cxs‐1, suppressed a clpP null mutation with respect to srf transcription, but did not overcome the block conferred by clpP on competence development and sporulation. Both cxs‐1 and cxs‐2 mutations map to the region of the rpoA gene encoding the RNA polymerase α C‐terminal domain (αCTD). The reconstruction of the cxs‐1 and cxs‐2 alleles of rpoA confirmed that these mutations confer the suppressor phenotype. These findings provide further support for the hypothesis that ClpX and ClpP might be intimately associated with transcription initiation in B. subtilis.
Elucidating the molecular mechanisms of bacterial virulence using non‐mammalian hosts
Authors: Mahajan‐Miklos Shalina , Rahme Laurence G. , Ausubel Frederick M.
Published: March 2003

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non‐mammalian hosts are used to screen directly for virulence‐attenuated mutants. The majority of PA14 mutants isolated using non‐mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host‐specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.
Enterococcus faecalis conjugative plasmid pAM373: complete nucleotide sequence and genetic analyses of sex pheromone response
Authors: De Boever Erika H. , Clewell Don B. , Fraser Claire M.
Published: March 2003

pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid‐free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone‐responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone‐binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface‐exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917–lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.
Completion of the hook–basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription
Authors: Karlinsey Joyce E. , Tanaka Shugo , Bettenworth Vera , Yamaguchi Shigeru , Boos Winfried , Aizawa Shin‐Ichi , Hughes Kelly T.
Published: March 2003

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline‐inducible promoter PtetA using the T‐POP transposon. Cells containing this construct are motile in the presence of tetracycline and non‐motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti‐sigma factor in the growth medium. This also coincides with the completion of hook–basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook–basal body structure.
A defined sequence within the 3′ UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation
Authors: Morozov Igor Y. , Martinez Marisa Galbis , Jones Meriel G. , Caddick Mark X.
Published: March 2003

Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.
The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37°C
Authors: Derré Isabelle , Rapoport Georges , Msadek Tarek
Published: March 2003

CtsR (class three stress gene repressor) negatively regulates the expression of class III heat shock genes (clpP, clpE and the clpC operon) by binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAA NAN A/GGTCAAA). CtsR‐dependent genes are expressed at a low level at 37°C and are strongly induced under heat shock conditions. We performed a structure/function analysis of the CtsR protein, which is highly conserved among low G+C Gram‐positive bacteria. Random chemical mutagenesis, in vitro cross‐linking, in vivo co‐expression of wild‐type and mutant forms of CtsR and the construction of chimeric proteins with the DNA‐binding domain of the λ CI repressor allowed us to identify three different functional domains within CtsR: a helix‐turn‐helix DNA‐binding domain, a dimerization domain and a putative heat‐sensing domain. We provide evidence suggesting that CtsR is active as a dimer. Transcriptional analysis of a clpP′–bgaB fusion and/or Western blotting experiments using antibodies directed against the CtsR protein indicate that ClpP and ClpX are involved in CtsR degradation at 37°C. This in turn leads to a low steady‐state level of CtsR within the cell, as CtsR negatively autoregulates its own synthesis. This is the first example of degradation of a repressor of stress response genes by the Clp ATP‐dependent protease.
Mid‐cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: co‐ordinating chromosome replication with cell division
Authors: Regamey A. , Harry E. J. , Wake R. G.
Published: March 2003

We have shown previously that, when spores of a thymine‐requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid‐cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the ‘premature’ mid‐cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid‐cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid‐cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid‐cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid‐cell Z ring assembly breaks down prematurely.
Two independent transcriptional units control the complex and simultaneous expression of the bmp paralogous chromosomal gene family in Borrelia burgdorferi
Authors: Dobrikova Elena Yu , Bugrysheva Julia , Cabello Felipe C.
Published: March 2003

The chromosomal paralogous gene family 36 encodes for four lipoproteins with high amino acid homology that are expressed in vivo in humans and animals and are immunogenic. Transcriptional analysis of the bmp gene cluster indicated that all four genes of this cluster are expressed in vitro and constitute two transcriptional units with a complex pattern of transcription, including alternative monocistronic and polycistronic messages. One unit consists of bmpD, whose transcription is coupled to the transcription of the ribosomal protein genes, rpsG and rpsL. The second unit includes bmpC, bmpA and bmpB. The simultaneous expression of the four bmp genes in Borrelia burgdorferi suggests that their gene products may have either different or complementary functions. Primer extension experiments identified promoters for bmpD, bmpC and bmpA, but not for bmpB. The concentration of gene‐specific mRNA paralleled its promoter homology to the Escherichia coliσ70 promoter. The linkage of bmpD expression to rpsL and rpsG suggests that the expression of this gene may be controlled by growth‐related global regulation mechanisms in B. burgdorferi. These results indicate that the bmp family constitutes a good model for the investigation of complex regulation of chromosomal gene expression in this bacteria.
Integrative module of the high‐pathogenicity island of Yersinia
Authors: Rakin A. , Noelting C. , Schropp P. , Heesemann J.
Published: March 2003

The high‐pathogenicity island of Yersinia pestis (Yps HPI) encodes virulence‐associated genes involved in siderophore yersiniabactin‐mediated iron uptake. The Yps HPI contains a P4‐type integrase (Int‐HPI), associated with the asn‐tRNA locus, and is flanked by 17 bp direct repeats. We constructed a minimal integrative module of the pathogenicity island carrying the reconstituted 266 bp attP (POP′) attachment site derived from putative attR and attL junctions of the Yps HPI and the functional int‐HPI gene from Y. pestis KUMA. The attP–int‐HPI module recombined efficiently, site specifically and RecA independently with the bacterial attB site present either in the chromosome (asn‐tDNA) or on a plasmid, with no preference for a certain asn‐tRNA gene. The excision of the integrated suicide plasmid carrying the integrative module, on the other hand, was a rare event and could be demonstrated only by polymerase chain reaction. Analysis of the 5′ terminus of the transcript for int‐HPI revealed that the integration of attP–int‐HPI was coupled with the replacement of the endogenous int‐HPI promoter, localized in the P′ part of the attP site, by the adjacent asn‐tRNA promoter. These results suggest that two alternative promoters control integration and excision of the HPI by its integrase.
Identification of a segment of DsbB essential for its respiration‐coupled oxidation
Authors: Kobayashi Taeko , Takahashi Yuki , Ito Koreaki
Published: March 2003

In the Escherichia coli protein disulphide bond formation pathway, membrane‐bound DsbB oxidizes periplasmic DsbA, the disulphide bond‐introducing enzyme. The Cys‐41–Val–Leu–Cys‐44 motif in the first periplasmic domain of DsbB is kept strongly oxidized by the respiratory function of the cell. We now show that the characteristic dithiothreitol resistance of the Cys‐41–Cys‐44 bond was retained even when the flanked Val–Leu combination was replaced by XX sequences from other oxidoreductases. Results of insertion mutagenesis showed that only the insertions (1–31 amino acids) in the region C‐terminally adjacent to the CXXC motif impaired the oxidized state of DsbB. Deletion of a single amino acid from this region also rendered DsbB reduced and inactive. However, single amino acid substitutions of the four residues flanked by CXXC and the transmembrane segment did not abolish the oxidation of DsbB. These results suggest that some physical property, such as distance of the CXXC motif from the membrane, is important for the respiration‐coupled oxidation of DsbB.
Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella
Authors: Figueroa‐Bossi Nara , Uzzau Sergio , Maloriol Danièla , Bossi Lionello
Published: March 2003

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy‐1 and Gifsy‐2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy‐2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III‐translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy‐1 and Gifsy‐2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels‐1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy‐3, which contains the phoP/phoQ‐activated pagJ gene and the gene for the secreted leucine‐rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEΦ. Most phage‐associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.
Precise amounts of a novel member of a phosphotransferase superfamily are essential for growth and normal morphology in Caulobacter crescentus
Authors: Fuchs T. , Wiget P. , Østerås M. , Jenal Urs
Published: March 2003

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10‐fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P‐type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site‐directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.