bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein
Authors: Christodoulides Myron , Everson J. Sylvia , Liu Bin L. , Lambden Paul R. , Watt Peter J. , Thomas Eric J. , Heckels John E.
Published: March 2003

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal–host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro‐inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL‐8 chemokine and reduced secretion of IL‐6 cytokine. Gonococcal challenge also caused increased secretion of TNF‐α cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non‐adherent bacteria may be involved in TNF‐α induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.
Molecular cloning and transcriptional regulation of ompT, a ToxR‐repressed gene in Vibrio cholerae
Authors: Li Caiyi C. , Crawford J. Adam , DiRita Victor J. , Kaper James B.
Published: March 2003

In pathogenic Vibrio cholerae, at least 17 genes are co‐ordinately regulated by ToxR. Most of these genes, including those that encode cholera toxin (CT), toxin co‐regulated pilus (TCP), accessory colonization factor (ACF) and OmpU, are positively regulated. OmpT is the only identified protein under negative regulation of ToxR. To understand the molecular mechanism by which ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR. Sequence analysis revealed that ompT encodes a predicted 35.8 kDa outer membrane porin of V. cholerae. Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon. Both primer extension analysis and promoter fusion studies showed that ToxR represses OmpT expression at the transcriptional level. Promoter fusion studies also suggest that cyclic AMP receptor protein (CRP) is involved in ompT activation. Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediates repression of ompT transcription by directly binding to an A/T‐rich region between −95 and −30 of the ompT promoter. To further understand how the interaction of ToxR with different promoters results in its function as an activator or repressor, we have also mapped the regions on the ctxAB and toxT promoters to which ToxR binds. The regions protected by ToxR on each of these promoters are all A/T rich and large in size, although they are positioned differently relative to each transcriptional start site.
The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases
Authors: De Bolle Xavier , Bayliss Christopher D. , Field Dawn , Van De Ven Tamsin , Saunders Nigel J. , Hood Derek W. , Moxon E. Richard
Published: March 2003

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5′‐AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild‐type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17–38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.
An Agrobacterium catalase is a virulence factor involved in tumorigenesis
Authors: Xu Xiu Q. , Pan Shen Q.
Published: March 2003

Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil‐borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T‐DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium–plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.
Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli
Authors: Steuber Julia , Schmid Claudia , Rufibach Michèle , Dimroth Peter
Published: March 2003

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate‐grown cells was 40 nmol mg−1 min−1 and was resistant to the uncoupler carbonyl‐cyanide m‐chlorophenylhydrazone (CCCP), but was inhibited by the complex I‐specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1–3 nmol mg−1 min−1), and rotenone was without effect.
Adaptation to the environment: Streptococcus pneumoniae, a paradigm for recombination‐mediated genetic plasticity?
Authors: Claverys Jean‐Pierre , Prudhomme Marc , Mortier‐Barrière Isabelle , Martin Bernard
Published: March 2003

Genetic plasticity plays a central role in the biology of the human pathogen Streptococcus pneumoniae. This is illustrated by the existence of at least 90 different capsular types (the polysaccharide capsule has an essential antiphagocytic function) as well as by the rapid emergence of penicillin‐resistant (PenR) pneumococcal isolates. Natural genetic transformation is believed to be essential for this genetic plasticity; capsular types can be switched by intraspecies transformation, whereas interspecies transformation is responsible for the appearance, in the PenR isolates, of mosaic pbp genes, which encode proteins with reduced affinity for penicillin. Data on the regulation of competence for transformation in S. pneumoniae, on the control of intra‐ and interspecies genetic exchange and on the shuffling and capture of exogenous sequences during transformation are reviewed. Possible links between transformation and changes in environmental conditions are discussed, and the adaptive ‘strategy’ deduced for S. pneumoniae is compared with that of Escherichia coli.
Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress
Authors: Krieger S. , Schwarz W. , Ariyanayagam M. R. , Fairlamb A. H. , Krauth‐Siegel R. L. , Clayton C.
Published: March 2003

In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione–glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline‐inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild‐type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild‐type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 μM) was observed compared with the wild type (EC50 = 223 μM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline‐regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.
The spliceosomal intron of the rolA gene of Agrobacterium rhizogenes is a prokaryotic promoter
Authors: Pandolfini T. , Storlazzi A. , Calabria E. , Defez R. , Spena A.
Published: March 2003

Agrobacterium rhizogenes transfers DNA (T‐DNA) from its Ri plasmid to plant cells. All T‐DNA genes are expressed in plant cells. The rolA gene is the only T‐DNA gene that contains an intron in the untranslated leader region of its mRNA. This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free‐living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5′ end and is reduced by mutations changing its sequence near the putative −10 region. The expression pattern of a chimeric reporter gene shows that, in free‐living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase. Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.
AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways
Authors: Römling Ute , Rohde Manfred , Olsén Arne , Normark Staffan , Reinköster Jürgen
Published: March 2003

The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long‐range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C‐terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long‐range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.
Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop
Authors: Wade Joseph T. , Belyaeva Tamara A. , Hyde Eva I. , Busby Stephen J. W.
Published: March 2003

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and −10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and −10 region. Parallels with the Escherichia coli araC–araBAD regulatory region are discussed.
Agr‐independent regulation of fibronectin‐binding protein(s) by the regulatory locus sar in Staphylococcus aureus
Authors: Wolz Christiane , Pöhlmann‐Dietze Petra , Steinhuber Andrea , Chien Yueh‐Tyng , Manna Adhar , Van Wamel Willem , Cheung Ambrose
Published: March 2003

Fibronectin‐binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site‐specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid‐phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar–agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA‐specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post‐transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.
Two novel proteins, PopB, which has functional nuclear localization signals, and PopC, which has a large leucine‐rich repeat domain, are secreted through the Hrp‐secretion apparatus of Ralstonia solanacearum
Authors: Guéneron Mareva , Timmers Antonius C. J. , Boucher Christian , Arlat Matthieu
Published: March 2003

The Ralstonia solanacearum hrp gene cluster codes for components of a type III secretion pathway necessary for the secretion of PopA1, a hypersensitive response‐like elicitor protein. In the present study, we show that several other Hrp‐secreted proteins can be detected by growing wild‐type bacteria in minimal medium in the presence of Congo red. Two of these proteins, PopB and PopC, are encoded by genes located downstream of popA and constitute an operon with popA. popABC mutants retain the wild‐type ability to cause disease in hosts and to elicit the hypersensitive response on non‐hosts. Expression of the popABC operon is controlled by the hrpB regulatory gene and is induced upon co‐culture with Arabidopsis cell suspensions. This plant cell‐specific induction depends on PrhA, a putative receptor for plant specific signal(s). The transcription of the popABC operon is not modified by the addition of Congo red to the growth medium and the intracellular pools of PopB and PopC are very similar in the absence or presence of Congo red. Preliminary data suggest that Congo red stabilizes secreted proteins in the extracellular medium. PopB is a 173‐amino‐acid‐basic protein that contains a functional bipartite nuclear localization signal. PopC is a 1024‐amino‐acid protein that carries 22 tandem leucine‐rich repeats (LRR). The LRR domain of this protein forms a consensus that perfectly matches the predicted eukaryotic cytoplasmic LRR consensus. We propose that PopB and PopC may be translocated into plant cells via the Hrp pathway.
Generation of a mature streptococcal cysteine proteinase is dependent on cell wall‐anchored M1 protein
Authors: Collin Mattias , Olsén Arne
Published: March 2003

In the present study, we have generated a mutant strain of Streptococcus pyogenes, MC25, which lacks M protein on its surface, and we demonstrate that this strain is unable to generate a mature 28 kDa cysteine proteinase. Furthermore, we show that S. pyogenes bacteria of M1 serotype are dependent on cell wall‐anchored M protein to cleave the secreted zymogen into a mature cysteine proteinase. We also show that MC25 secretes a 40 kDa zymogen, having a conformation different from that secreted by wild‐type bacteria. We provide data showing that the cleavage site is not blocked but, presumably, the active site is. This suggests that M protein, when anchored to the cell wall, is involved in the unfolding of the zymogen and generation of a mature cysteine proteinase that can be activated under reducing conditions. Our data add new aspects to the interaction between two important virulence factors of S. pyogenes, the streptococcal cysteine proteinase and M protein.
Proteolysis of SpoIVB is a critical determinant in signalling of Pro‐σK processing in Bacillus subtilis
Authors: Wakeley Philip R. , Dorazi Robert , Hoa Ngo Thi , Bowyer John R. , Cutting Simon M.
Published: March 2003

SpoIVB is essential for intercompartmental signalling in the σK‐checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro‐σK to its mature and active form σK. We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self‐cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro‐σK processing and a yet, uncharacterized, second function which is essential for the formation of heat‐resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.
Evidence for the genetic interaction between the actin‐binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae
Authors: Roumanie Olivier , Peypouquet Marie France , Bonneu Marc , Thoraval Didier , Doignon François , Crouzet Marc
Published: March 2003

The non‐essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH–coiled coil–RhoGAP domain organization, showed a patch‐like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1Δ and vrp1Δ mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1–VRP1 co‐lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton‐linked genes were also studied. These and other well‐established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton‐based activities, in co‐operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.
Identification and characterization of SarH1, a new global regulator of virulence gene expression in Staphylococcus aureus
Authors: Tegmark Karin , Karlsson Anna , Arvidson Staffan
Published: March 2003

The global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) have been reported to be both activators and repressors of virulence gene expression in Staphylococcus aureus. How the effector of the agr system, RNAIII, interacts with target gene promoters is unknown. SarA, on the other hand, is a DNA‐binding protein, which binds to conserved DNA motifs immediately upstream of both positively and negatively regulated promoters. Here, we searched for additional regulators that could explain the differential effects of RNAIII and SarA. Four differently regulated genes (hla, alpha‐toxin; hld, RNAIII; spa, protein A; ssp, serine protease) were analysed for binding of potential regulatory proteins to the corresponding promoter DNA fragments, linked to magnetic beads. One protein (29 kDa), with affinity for all four promoters, showed a high degree of similarity to SarA and was named SarH1 (Sar homologue 1). Expression of sarH1 was strongly repressed by sarA and agr. Analysis of hla, hld, ssp and spa mRNAs in sarH1, sarA and agr mutants, and in sarA/sarH1 and agr/sarH1 double mutants, revealed that sarH1 has a strong repressive effect on hla and an activating effect on spa transcription. SDS–PAGE analysis of secreted proteins from the different mutants showed that the production of several other exoproteins was affected by sarH1. In conclusion, we show that both the agr‐dependent suppression of protein A production and the sarA‐dependent stimulation of alpha‐toxin production is mediated via a new regulator, SarH1, which belongs to a family of Sar homologues.
Targeted disruption of the glycosylphosphatidylinositol‐anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice
Authors: Dzierszinski Florence , Mortuaire Marlène , Cesbron‐Delauw Marie France , Tomavo Stanislas
Published: March 2003

The protozoan parasite Toxoplasma gondii is able to invade a broad range of cells within its mammalian hosts through mechanisms that are not yet fully understood. Several glycosylphosphatidylinositol‐anchored antigens found in the parasite membrane are considered as major determinants in the critical interactions with the host cell. We have discovered that two of these surface antigens, SAG1 and SAG3, share significant identity, with considerable similarities in structure, suggesting an overall conserved topology. To investigate their physiological roles further, we have generated T. gondii mutants deficient in SAG3 through gene disruption. The disrupted strains display at least a twofold reduction in host cell invasion when compared with wild‐type parasites. This correlated with a similar decrease in host cell adhesion in the SAG3 null mutants. Importantly, the null SAG3 mutants show attenuated infectivity, with a markedly reduced capacity to cause mortality in mice, whereas both wild‐type and complemented mutants that re‐expressed SAG3 were lethal at the same doses. Taken together, our results indicate that SAG3 is one member of the redundant system of T. gondii receptors that act as ligands mediating host cell recognition and attachment.
A role for the fibrinogen‐binding regions of streptococcal M proteins in phagocytosis resistance
Authors: Ringdahl Ulrika , Svensson Henrik G. , Kotarsky Heike , Gustafsson Maria , Weineisen Maria , Sjöbring Ulf
Published: March 2003

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type‐specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen‐binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen‐binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.
Regulation of the CYB2 gene expression: transcriptional co‐ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors
Authors: Ramil Elvira , Agrimonti Catarina , Shechter Evelyne , Gervais Michel , Guiard Bernard
Published: March 2003

Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme l‐(+)‐lactate–cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA–protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co‐regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.
DNase I homologous residues in CdtB are critical for cytolethal distending toxin‐mediated cell cycle arrest
Authors: Elwell Cherilyn A. , Dreyfus Lawrence A.
Published: March 2003

Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard blast searches, a position‐specific iterated (psi) blast search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position‐specific homology to type I mammalian DNases. The psiblast sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild‐type CdtA and CdtC polypeptides. Mutation in four of the five DNase‐specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild‐type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT‐induced cell cycle arrest.
Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response
Authors: Raivio Tracy L. , Laird Michael W. , Joly John C. , Silhavy Thomas J.
Published: March 2003

The Cpx envelope stress response of Escherichia coli is controlled by a two‐component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP–CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the σE stress response. Because the σE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.
ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation
Authors: Zhang Xiaohui , Hulett F. Marion
Published: March 2003

A two‐component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A‐containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD‐controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the −35 promoter region and one (A3) downstream of the −10 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter–lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD∼P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter–lacZ fusion constructs showed that DNA containing ResD‐binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.
The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri
Authors: Buchrieser Carmen , Glaser Philippe , Rusniok Christophe , Nedjari Hafed , D'Hauteville Hélène , Kunst Frank , Sansonetti Philippe , Parsot Claude
Published: March 2003

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi–Spa type III secretion apparatus and the secreted IpaA–D proteins, all of which are encoded by a virulence plasmid. In wild‐type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N‐terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.
Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation
Authors: Alugupalli Kishore R. , Michelson Alan D. , Barnard Marc R. , Robbins Douglas , Coburn Jenifer , Baker Elizabeth K. , Ginsberg Mark H. , Schwan Tom G. , Leong John M.
Published: March 2003

Borrelia hermsii, a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro. Platelet binding was promoted by the platelet integrin αIIbβ3: the bacterium bound to purified integrin αIIbβ3, and bacterial binding to platelets was diminished by αIIbβ3 antagonists or by a genetic defect in this integrin. Integrin αIIbβ3 undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of αIIbβ3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin αIIbβ3 activation, as indicated by the expression of the platelet activation marker P‐selectin and integrin αIIbβ3 in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I2, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high‐level bacterial attachment.
Type 1 fimbriae deliver an LPS‐ and TLR4‐dependent activation signal to CD14‐negative cells
Authors: Hedlund Maria , Frendéus Björn , Wachtler Caroline , Hang Long , Fischer Hans , Svanborg Catharina
Published: March 2003

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae‐dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll‐like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS‐ and TLR4‐dependent signal to the CD14‐negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS‐dependent response in those cells. A similar LPS‐ and fimbriae‐dependent response was observed in the urinary tract of TLR4‐proficient mice, but not in TLR4‐defective mice. The moderate inflammatory response in the TLR4‐defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14‐negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host ‘sees’ LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.
Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H‐NS‐dependent repression
Authors: Bustamante Vı´ctor H. , Santana Francisco J. , Calva Edmundo , Puente José Luis
Published: March 2003

Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone‐like protein H‐NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37°C (e.g. 40°C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37°C (e.g. 25°C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H‐NS‐mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H‐NS–nucleoprotein complex.
Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans
Authors: Cox Gary M. , McDade Henry C. , Chen Sharon C. A. , Tucker Stephanie C. , Gottfredsson Magnus , Wright Lesley C. , Sorrell Tania C. , Leidich Steven D. , Casadevall Arturo , Ghannoum Mahmoud A. , Perfect John R.
Published: March 2003

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase‐encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild‐type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37°C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild‐type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage‐like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.
Functional characterization of the α‐glucoside transporter Sut1p from Schizosaccharomyces pombe, the first fungal homologue of plant sucrose transporters
Authors: Reinders Anke , Ward John M.
Published: March 2003

Disaccharide transporters have not previously been identified in Schizosaccharomyces pombe. This is in contrast to Saccharomyces cerevisiae in which several maltose permeases belonging to the sugar porter (SP) family have been characterized. Here we report that a novel S. pombe gene, sut1+, encodes a proton‐coupled disaccharide uptake transporter in the glycoside–pentoside–hexuronide (GPH):cation symporter family. Previously, members of the GPH family were restricted to bacteria and plants. The closest homologues of sut1+ are the sucrose uptake transporters (SUTs) from higher plants that transport sucrose with a higher affinity than maltose. The transport function of Sut1p was analysed by expression in S. cerevisiae. Sut1p was found to transport maltose with a Km of 6.5 ± 0.4 mM and sucrose with a Km of 36.3 ± 9.7 mM. Therefore, the substrate specificity of Sut1p from S. pombe is different from that of its plant homologues. Glucose repression of sut1+ at the transcriptional level is also consistent with a physiological function for Sut1p in maltose uptake. These results indicate that, unlike S. cerevisiae, S. pombe utilizes maltose transporters derived from a common ancestor with the plant SUTs.
The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin‐binding and cysteine protease activity
Authors: Hytönen Jukka , Haataja Sauli , Gerlach Dieter , Podbielski Andreas , Finne Jukka
Published: March 2003

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein‐binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin‐binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.
Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida
Authors: Duque Estrella , Segura Ana , Mosqueda Gilberto , Ramos Juan L.
Published: March 2003

Pseudomonas putida DOT‐T1E grows on a water–toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR‐deficient background, expression from the ttgA promoter increased about 20‐fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild‐type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp‐like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp‐like protein is a global regulatory protein involved in the solvent‐tolerant response of this strain.
An essential dimeric membrane protein of trypanosome glycosomes
Authors: Maier Alexander , Lorenz Patrick , Voncken Frank , Clayton Christine
Published: March 2003

Kinetoplastid parasites compartmentalize the first seven enzymes of glycolysis in a peroxisome‐like microbody, the glycosome. Genes encoding the most abundant protein of the glycosomal membrane, GIM5, have been cloned and the protein characterized. Two genes, GIM5A and GIM5B, encode 26 kDa proteins. Although many microbody membrane proteins are conserved in evolution, the only homologues of GIM5 in the available databases are from the closely related kinetoplastids Trypanosoma cruzi and Leishmania. The N‐ and C‐termini are conserved between the two genes, and between species, and are oriented towards the cytosol. They are separated by a short loop that is located between two transmembrane domains and shows almost no sequence conservation. This suggests that the N‐ and C‐terminal domains are more important for function. GIM5 forms dimers in vivo. Overexpression of GIM5B inhibits growth, whereas depletion of GIM5 to below 10% of wild‐type levels is very rapidly lethal. This novel organellar membrane protein is therefore essential for bloodstream trypanosome survival.