bluuebunny/crossref_metadata_2025 huggingface dataset:
Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli
Authors: Xia Yan , Forsman Kristina , Jass Jana , Uhlin Bernt Eric
Published: March 2003

Transcriptional regulation of the pap genes, which encode fimbrial adhesins in uropathogenic Escherichia coli, depends on an upstream activating region. This region contains binding sites for a transcription factor, PapB, which is a member of a growing family of putative regulatory proteins found in several virulence‐associated fimbrial gene systems. To assess the nature of the PapB binding sites, we studied different naturally occurring variants and a number of in vitro constructed mutant binding sites. DNase I footprinting analysis and visualization of the PapB–DNA complex by atomic force microscopy showed that the protein occupied a DNA region of more than 50 bp. Purified PapB protein was shown to recognize a motif including a 9 bp repeat sequence containing T/A triplets at a conserved position. PapB binding was affected by distamycin, and the results were consistent with the possibility that the binding to DNA occurred through minor groove interaction. From these analyses and estimation of the relative number of PapB proteins per binding site, we suggest that PapB binds the DNA in an oligomeric fashion and may function as an architectural factor in the transcriptional control of adhesin expression.
Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair
Authors: Mehr Ian J. , Seifert H. Steven
Published: March 2003

Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA‐dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high‐frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF‐like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.
Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two‐hybrid system
Authors: Kornacker M. G. , Remsburg B. , Menzel R.
Published: March 2003

The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC‐dependent gene activation by LexA. Repression also requires a LexA operator half‐site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator‐bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co‐operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli‐based two‐hybrid system.
A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis
Authors: Day James B. , Plano Gregory V.
Published: March 2003

Human pathogenic Yersinia resist host defences, in part through the expression and delivery of a set of plasmid‐encoded virulence proteins termed Yops. A number of these Yops are exported from the bacteria directly into the cytoplasm of their eukaryotic host's cells upon contact with these cells. The secreted YopN protein (also known as LcrE) is required to block Yop secretion in the presence of calcium in vitro or before contact with a eukaryotic cell in vivo. In this study, we characterize the role of the tyeA, sycN and yscB gene products in the regulation of Yop secretion in Yersinia pestis. Mutants specifically defective in the expression of TyeA, SycN or YscB were no longer able to block Yop secretion in the presence of calcium. In addition, the secretion of YopN was specifically reduced in both the sycN and the yscB deletion mutants. Protein cross‐linking and immunoprecipitation studies in conjunction with yeast two‐hybrid analyses showed that SycN and YscB interact with one another to form a SycN/YscB complex. Yeast three‐hybrid analyses demonstrated that the SycN/YscB complex, but not SycN or YscB alone, specifically associates with YopN. SycN and YscB share amino acid sequence similarity and structural similarities with the specific Yop chaperones SycE and SycH. Together, these results indicate that a complex composed of SycN and YscB functions as a specific chaperone for YopN in Y. pestis.
Differentiation‐associated surface antigen variation in the ancient eukaryote Giardia lamblia
Authors: Svärd Staffan G. , Meng Tze‐Chiang , Hetsko Michael L. , McCaffery J. Michael , Gillin Frances D.
Published: March 2003

Encystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome‐like vacuoles. As short‐range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation–excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.
In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene
Authors: Jacobi C. A. , Roggenkamp A. , Rakin A. , Zumbihl R. , Leitritz L. , Heesemann J.
Published: March 2003

The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE–GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co‐expressed in Y. enterocolitica (i) on a low‐copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co‐integration by homologous recombination of a yopE–gfp‐carrying suicide plasmid into pYV08 (co‐integrate configuration). After 30 min of infection of HEp‐2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE–GFP into HEp‐2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp‐2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE–GFP from selectively solubilized HEp‐2 cells. The co‐translocation of other Yops was not significantly impaired by YopE–GFP as shown by YopH/YopE‐mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE–gfp expression (in trans as well as in the co‐integrate configuration) in the HEp‐2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE–LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.
D‐Erythroascorbic acid is an important antioxidant molecule inSaccharomyces cerevisiae
Authors: Huh Won‐Ki , Lee Byung‐Hoon , Kim Seong‐Tae , Kim Yeon‐Ran , Rhie Gi‐Eun , Baek Yong‐Woon , Hwang Cheol‐Sang , Lee Jung‐Shin , Kang Sa‐Ouk
Published: March 2003

D‐Arabinono‐1,4‐lactone oxidase catalysing the final step ofD‐erythroascorbic acid biosynthesis was purified from the mitochondrial fraction ofSaccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as theALO1gene encoding the enzyme. The ORF ofALO1encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59 493 Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that ofL‐gulono‐1,4‐lactone oxidase from rat andL‐galactono‐1,4‐lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of theALO1gene was 1.8 kb in size. In thealo1mutants,D‐erythroascorbic acid and the activity ofD‐arabinono‐1,4‐lactone oxidase could not be detected. The intracellular concentration ofD‐erythroascorbic acid and the enzyme activity increased up to 6.9‐fold and 7.3‐fold, respectively, in the transformant cells carryingALO1in multicopy plasmid. Thealo1mutants showed increased sensitivity towards oxidative stress, but overexpression ofALO1made the cells more resistant to oxidative stress.
Germination of Bacillus anthracis spores within alveolar macrophages
Authors: Guidi‐Rontani Chantal , Weber‐Levy Martine , Labruyère Elisabeth , Mock Michèle
Published: March 2003

The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage‐like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans‐activator, AtxA, were expressed within the macrophages after germination.
prhJ and hrpG, two new components of the plant signal‐dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum
Authors: Brito Belen , Marenda Marc , Barberis Patrick , Boucher Christian , Genin Stéphane
Published: March 2003

hrp gene expression in the phytopathogenic bacterium Ralstonia solanacearum GMI1000 is induced through the HrpB regulator in minimal medium and upon co‐culture with plant cell suspensions. The putative outer membrane protein PrhA is specifically involved in hrp gene activation in the presence of plant cells and has been proposed to be a receptor of a plant‐dependent signal transduction pathway. Here, we report on the identification of two regulatory genes, hrpG and prhJlocated at the right‐hand end of the hrp gene cluster, that are required for full pathogenicity. HrpG belongs to the OmpR subclass of two‐component response regulators and is homologous to HrpG, the activator of hrp genes in Xanthomonas campestris pv. vesicatoria. PrhJ is a novel hrp regulatory protein, sharing homology with the LuxR/UhpA family of transcriptional activators. As for HrpG of X. c. pv. vesicatoria, HrpG is required for hrp gene expression in minimal medium, but, in addition, we show that it also controls hrpB gene activation upon co‐culture with Arabidopsis thaliana and tomato cell suspensions. In contrast, PrhJ is specifically involved in hrp gene expression in the presence of plant cells. hrpG and prhJ gene transcription is plant cell inducible through the PrhA‐dependent pathway. From these results, we propose a regulatory cascade in which plant cell signal(s) sensed by PrhA are transduced to the prhJ gene, whose predicted product controls hrpG gene expression. HrpG then activates the hrpB regulatory gene, and, subsequently, the remaining hrp transcriptional units in all known inducing conditions.
Multiple independent regulatory pathways control UBI4 expression after heat shock in Saccharomyces cerevisiae
Authors: Simon John R. , Treger Janet M. , McEntee Kevin
Published: March 2003

Transcription of the polyubiquitin gene UBI4 of Saccharomyces cerevisiae is strongly induced by a variety of environmental stresses, such as heat shock, nutrient depletion and exposure to DNA‐damaging agents. This transcriptional response of UBI4 is likely to be the primary mechanism for increasing the pool of ubiquitin for degradation of stress‐damaged proteins. Deletion and promoter fusion studies of the 5′ regulatory sequences indicated that two different elements, heat shock elements (HSEs) and stress response element (STREs), contributed independently to heat shock regulation of the UBI4 gene. In the absence of HSEs, STRE sequences localized to the intervals −264 to −238 and −215 to −183 were needed for stress control of transcription after heat shock. Site‐directed mutagenesis of the STRE (AG4) at −252 to −248 abolished heat shock induction of UBI4 transcription. Northern analysis demonstrated that cells containing either a temperature‐sensitive HSF or non‐functional Msn2p/Msn4p transcription factors induced high levels of UBI4 transcripts after heat shock. In cells deficient in both heat stress pathways, heat‐induced UBI4 transcript levels were considerably lower but not abolished, suggesting a role for another factor(s) in stress control of its expression.
Molecular genetic basis for the variable expression of Lewis Y antigen in Helicobacter pylori : analysis of the α(1,2) fucosyltransferase gene
Authors: Wang Ge , Rasko David A. , Sherburne Richard , Taylor Diane E.
Published: March 2003

Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of α(1,3) and α(1,2) fucosyltransferases (FucTs). Here, we report the molecular cloning and characterization of genes encoding H. pyloriα(1,2) FucT (Hp fucT2 ) from various H. pylori strains. We constructed Hp fucT2 knock‐out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme‐linked immunosorbent assay (ELISA) and immunoelectron microscopy. The Hp fucT2 gene contains a hypermutable sequence [poly (C) and TAA repeats], which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full‐length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full‐length protein. The production of the full‐length protein by the 26695 fucT2 gene could be attributed to translational −1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present. Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype. In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by H. pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved.
Recruitment of ZipA to the division site by interaction with FtsZ
Authors: Liu Zhan , Mukherjee Amit , Lutkenhaus Joe
Published: March 2003

ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle. As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring. In this study, we have investigated the requirements for ZipA localization to the division site. ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ. Consistent with this, apparently normal Z rings are formed in the absence of ZipA. The interaction between FtsZ and ZipA occurs through their carboxy‐terminal domains. Although a MalE–ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments. These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.
Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature‐tagged transposon mutagenesis
Authors: Darwin Andrew J. , Miller Virginia L.
Published: March 2003

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature‐tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild‐type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O‐antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.
Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells
Authors: Hartland Elizabeth L. , Batchelor Miranda , Delahay Robin M. , Hale Christine , Matthews Stephen , Dougan Gordon , Knutton Stuart , Connerton Ian , Frankel Gad
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin‐binding domain of Tir to a central 107‐amino‐acid region, designated Tir‐M. We provide evidence that both the amino‐ and carboxy‐termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir. This Tir‐independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin‐like module residing at the carboxy‐terminus of the intimin polypeptide. Using the yeast two‐hybrid system and gel overlays, we show that intimin can bind both Tir and Tir‐M even when the lectin‐like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectin‐like manner with a host cell intimin receptor.
The C‐terminus of eRF1 defines a functionally important domain for translation termination inSaccharomyces cerevisiae
Authors: Eurwilaichitr Lily , Graves Fiona M. , Stansfield Ian , Tuite Mick F.
Published: March 2003

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C‐terminal deletion analysis of eRF1 from the yeastSaccharomyces cerevisiae, we show that the extreme C‐terminus of this 437‐amino‐acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C‐terminal 32 amino acids is not viable, whereas deletion of the C‐terminal 19 amino acids is viable but shows a termination defectin vivocausing an enhancement of nonsense suppression. Using a combination of two‐hybrid analysis andin vitrobinding studies, we demonstrate that deletions encompassing the C‐terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C‐terminally truncated eRF1 still bind the ribosome, suggesting that the C‐terminus does not constitute a ribosome‐binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C‐terminus, although our deletion studies suggest that it is sequences C‐terminal to this region that are functionally important.
Substrate‐specific binding of hook‐associated proteins by FlgN and FliT, putative chaperones for flagellum assembly
Authors: Fraser Gillian M. , Bennett John C. Q. , Hughes Colin
Published: March 2003

During flagellum assembly by motile enterobacteria, flagellar axial proteins destined for polymerization into the cell surface structure are thought to be exported through the 25–30 Å flagellum central channel as partially unfolded monomers. How are premature folding and oligomerization in the cytosol prevented? We have shown previously using hyperflagellated Proteus mirabilis and a motile but non‐swarming flgN transposon mutant that the apparently cytosolic 16.5 kDa flagellar protein FlgN facilitates efficient flagellum filament assembly. Here, we investigate further whether FlgN, predicted to contain a C‐terminal amphipathic helix typical of type III export chaperones, acts as a chaperone for axial proteins. Incubation of soluble radiolabelled FlgN from Salmonella typhimurium with nitrocellulose‐immobilized cell lysates of wild‐type S. typhimurium and a non‐flagellate class 1 flhDC mutant indicated that FlgN binds to flagellar proteins. Identical affinity blot analysis of culture supernatants from the wild‐type and flhDC, flgI, flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook‐associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. Analysis of axial proteins secreted into the culture medium by the original P. mirabilis flgN mutant demonstrated that export of FlgK and FlgL was specifically reduced, with concomitant increased release of unpolymerized flagellin (FliC), the immediately distal component of the flagellum. These data suggest that FlgN functions as an export chaperone for FlgK and FlgL. Parallel experiments showed that FliT, a similarly small (14 kDa), potentially helical flagellar protein, binds specifically to the flagellar filament cap protein, FliD (HAP2), indicating that it too might be an export chaperone. Flagellar axial proteins all contain amphipathic helices at their termini. Removal of the HAP C‐terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C‐termini were specifically bound by FlgN and FliT. We suggest that FlgN and FliT are substrate‐specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export.
The product of the SNF2/SWI2 paralogue INO80 of Saccharomyces cerevisiae required for efficient expression of various yeast structural genes is part of a high‐molecular‐weight protein complex
Authors: Ebbert Ronald , Birkmann Alexander , Schüller Hans‐Joachim
Published: March 2003

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are activated by the Ino2p/Ino4p transcription factor that binds to ICRE promoter motifs and mediates maximal gene expression in the absence of inositol. We identified the ino80 mutation causing inositol auxotrophy as a result of a defect in ICRE‐dependent gene activation. The product of the corresponding wild‐type gene INO80 (= YGL150C) shows significant similarity to the Snf2p family of DNA‐dependent ATPases. Nevertheless, SNF2 in increased gene dosage did not suppress ino80 mutant phenotypes. Mutation of the Ino80p lysine residue corresponding to the NTP binding site of Snf2p led to a non‐functional protein. In ino80 null mutants, gene activation mediated by an ICRE decreased to 16% of the wild‐type level. Maximal expression of PHO5, GAL1, CYC1 and ICL1 was also significantly reduced. Thus, Ino80p affects several transcription factors involved in unrelated pathways. As demonstrated by gel filtration, Ino80p is part of a high‐molecular‐weight complex of more than 1 MDa. Similar to what was found for Snf2p, the Ino80p‐containing complex may influence the transcriptional level of several unrelated structural genes by functioning as an ATPase that possibly acts on chromatin.
Sequential gene disruption in Candida albicans by FLP‐mediated site‐specific recombination
Authors: Morschhäuser Joachim , Michel Sonja , Staib Peter
Published: March 2003

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP‐mediated, site‐specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P–FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP‐mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.
The V‐antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation
Authors: Pettersson Jonas , Holmström Anna , Hill Jim , Leary Sophie , Frithz‐Lindsten Elisabet , Von Euler‐Matell Anne , Carlsson Eva , Titball Richard , Forsberg Åke , Wolf‐Watz Hans
Published: March 2003

Type III‐mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic YersiniaLcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti‐LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV‐induced protective immunity.
PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis
Authors: Agaisse Hervé , Gominet Myriam , Økstad Ole Andreas , Kolstø Anne‐Brit , Lereclus Didier
Published: March 2003

Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis ) are well‐known pathogens of mammals (B. anthracis and B. cereus ) and insects (B. thuringiensis ). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol‐specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR‐regulated expression. These PlcR‐regulated genes encode degradative enzymes, cell‐surface proteins and enterotoxins. The PlcR‐regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR‐regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus plcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis‐based biopesticides.
The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T‐rich region
Authors: Sohaskey Charles D. , Zückert Wolfram R. , Barbour Alan G.
Published: March 2003

OspA and B proteins of Borrelia burgdorferi and Vmp proteins of Borrelia hermsii are abundant outer membrane lipoproteins, whose expression varies with the environment. The genes for these proteins have the ‘−35′ and ‘−10’ elements of a σ70‐type promoter. Deletions of the promoters for these genes were analysed with a chloramphenicol acetyltransferase (CAT) reporter gene and plasmid constructs that were stably maintained in Escherichia coli or transiently transfected into B. burgdorferi. Reporter expression was measured as susceptibility of transformed E. coli cells to chloramphenicol and the CAT activity of E. coli and B. burgdorferi lysates in vitro. Presence of the ‘−10’ element was essential for full activity in both B. burgdorferi and E. coli. Upstream of the ‘−35′ elements of the ospAB and vmp promoters were tracts with Ts in 16 of 20 positions for B. burgdorferi and 18 of 20 positions for B. hermsii. Deletion of the T‐rich region from the ospAB or vmp promoter caused a greater reduction of CAT activity in B. burgdorferi than in E. coli. The findings indicate that ospAB and vmp promoters are extended promoters with two parts: (i) a core region containing typical ‘−35′ and ‘−10’ elements and (ii) a unique T‐rich region.
Selection of the midcell division site in Bacillus subtilis through MinD‐dependent polar localization and activation of MinC
Authors: Marston Adele L. , Errington Jeffery
Published: March 2003

Bacterial cell division commences with the assembly of the tubulin‐like protein, FtsZ, at midcell to form a ring. Division site selection in rod‐shaped bacteria is mediated by MinC and MinD, which form a division inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD. The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites. By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild‐type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.
The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole
Authors: González‐Zorn Bruno , Domínguez‐Bernal Gustavo , Suárez Mónica , Ripio María‐Teresa , Vega Yolanda , Novella Susana , Vázquez‐Boland José‐Antonio
Published: March 2003

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel‐shaped co‐operative haemolytic (‘CAMP‐like’) reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (β‐toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low‐stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock‐out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel‐shaped CAMP‐like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial‐like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane‐damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane‐damaging activity that, together with PlcA and PlcB, may act in concert with the pore‐forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5′ end of smcL is contiguous with the internalin locus i‐inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.
Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE
Authors: Hu Zonglin , Lutkenhaus Joe
Published: March 2003

Placement of the Z ring at midcell in Escherichia coli is assured by the action of the min system, which blocks usage of potential division sites that exist at the cell poles. This activity of min is achieved through the action of an inhibitor of division, MinC, that is activated by MinD and topologically regulated by MinE. In this study, we have used a functional GFP–MinC fusion to monitor the location of MinC. We find that GFP–MinC is a cytoplasmic protein in the absence of the other Min proteins. The addition of MinD, a peripheral membrane protein that interacts with MinC, results in GFP–MinC appearing on the membrane. In the presence of both MinD and MinE, GFP–MinC oscillates rapidly between the halves of the cell. Thus, MinC is positioned by the other Min products, but in a dynamic manner so that it is in position to inhibit Z ring assembly away from midcell.
Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two‐component system rcsC–rcsB
Authors: Carballès Fabrice , Bertrand Claire , Bouché Jean‐Pierre , Cam Kaymeuang
Published: March 2003

Genes rcsC and rcsB form a two‐component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB‐dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co‐regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.
Histidine kinases: diversity of domain organization
Authors: Dutta Rinku , Qin Ling , Inouye Masayori
Published: March 2003

Histidine kinases play a major role in signal transduction in prokaryotes for the cellular adaptation to environmental conditions and stresses. Recent progress in the three‐dimensional structure determination of two representative members of histidine kinases, EnvZ (class I) and CheA (class II), has revealed common structural features, as well as a kinase catalytic motif topologically similar to those of the ATP‐binding domains of a few ATPases. They have also disclosed that there are significant differences in domain organization between class I and II histidine kinases, possibly reflecting their distinct locations, functions and regulatory mechanisms. In spite of this diversity, both class I and II histidine kinases use similar four‐helix bundle motifs to relay phosphoryl groups from ATP to regulatory domains of response regulators. The previously known so‐called transmitter domain of histidine kinase is further dissected into two domains: a CA (Catalytic ATP‐binding) domain and a DHp (Dimerization Histidine phosphotransfer) domain for class I, or a CA domain and an HPt (Histidine‐containing Phosphotransfer) domain for class II histidine kinases. From a comparative analysis of the CA domains of EnvZ, CheA and their ATPase homologues, the core elements of the CA domain have been derived. The apparent resemblance between DHp and HPt domains is only superficial, and significant differences between them are discussed.
Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of Gram‐positive bacteria
Authors: Jonquières Renaud , Bierne Hélène , Fiedler Franz , Gounon Pierre , Cossart Pascale
Published: March 2003

InlB is a Listeria monocytogenes protein that is sufficient to promote entry in a variety of mammalian cells. The last 232‐amino‐acid domain (Csa) of InlB has been shown to mediate attachment on the listerial surface, although its sequence does not suggest any known mechanism of association to the bacterial surface. InlB is present both on the bacterial surface and in culture supernatants. As has been recently demonstrated, both forms of InlB, soluble and surface‐bound, can trigger signalling in host cells. To elucidate the specific role of each of the two forms, it was important to understand how InlB associates with the bacterial surface. Using microscopy, we find evidence that InlB is partially buried in the cell wall layer, and using fractionation experiments we demonstrate that InlB associates with the bacterial cytoplasmic membrane. Moreover, using purified lipoteichoic acid (LTA) and the three polypeptides InlB, Csa, or InlBΔCsa (InlB lacking the last 232 amino acids), we demonstrate that LTA is a ligand for the Csa domain of InlB. These results provide the first evidence of an interaction between lipoteichoic acids and a bacterial protein involved in adhesion and signalling, and highlight a new mechanism of protein association on the surface of Gram‐positive bacteria.
Extensive interplasmidic duplications change the virulence phenotype of the relapsing fever agent Borrelia turicatae
Authors: Penningon Pamela M. , Cadavid Diego , Bunikis Jonas , Norris Steven J. , Barbour Alan G.
Published: March 2003

The relapsing fever agent Borrelia turicatae has two antigenically distinct serotypes, A and B, which differ in their variable small proteins (Vsps) and in their degree of virulence and neurotropism in mice. Each Vsp gene (vspA or vspB) had an expression‐linked copy that was unique to the serotype expressing it. This was located on one linear plasmid, which was defined by the upstream sequence. The archived copies of vspA and vspB were each located on different linear plasmids that were the same in both serotypes. In this feature, the mechanism of antigenic variation is similar to that of another relapsing fever agent, B. hermsii. However, in other features, the mechanisms of the two organisms differ. The expressed and archived loci for vspA and vspB of B. turicatae were near the centre of linear plasmids instead of near the telomeres. The vspA and vspB expression loci were duplicate copies of their respective silent loci: from the vsp itself to at least 13–14 kb downstream. Despite the extensive interplasmidic duplications and the internal position of the expression locus, the only detectable difference between serotypes A and B was in whether they expressed VspA or VspB.
Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes : implications for phage evolution
Authors: Loessner Martin J. , Inman Ross B. , Lauer Peter , Calendar Richard
Published: March 2003

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double‐stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross‐linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy‐two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle‐specific manner into at least three major transcriptional units. N‐terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control‐associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site‐specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.
CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes
Authors: Nair Shamila , Derré Isabelle , Msadek Tarek , Gaillot Olivier , Berche Patrick
Published: March 2003

Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR‐deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42°C), but its level of virulence in the mouse was not affected. The virulence of a wild‐type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen.