bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di‐rhamnolipid biosynthesis
Authors: Rahim Rahim , Ochsner Urs A. , Olvera Clarita , Graninger Michael , Messner Paul , Lam Joseph S. , Soberón‐Chávez Gloria
Published: March 2003

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso‐active glycolipids containing one (mono‐rhamnolipid) or two (di‐rhamnolipid) l‐rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono‐rhamnolipid from dTDP‐l‐rhamnose and β‐hydroxydecanoyl‐β‐hydroxydecanoate, whereas di‐rhamnolipid is produced from mono‐rhamnolipid and dTDP‐l‐rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di‐rhamnolipid (l‐rhamnose‐l‐rhamnose‐β‐hydroxydecanoyl‐β‐hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of l‐rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII‐2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di‐rhamnolipid, whereas mono‐rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1‐rhlA) was incapable of producing both mono‐ and di‐rhamnolipid. Complementation of mutant RTII‐2 with plasmid pRTII‐26 containing rhlC restored the level of di‐rhamnolipid production in the recombinant to a level similar to that of the wild‐type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A σ54‐type promoter for the PA1131–rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131–rhlC operon was dependent on the P. aeruginosa rhl quorum‐sensing system, and a well‐conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di‐rhamnolipid.
Molecular characterization of the surface layer proteins from Clostridium difficile
Authors: Calabi Emanuela , Ward Steven , Wren Brendan , Paxton Thanai , Panico Maria , Morris Howard , Dell Anne , Dougan Gordon , Fairweather Neil
Published: March 2003

Many bacteria express a surface‐exposed proteinaceous layer, termed the S‐layer, which forms a regular two‐dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S‐layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S‐layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post‐translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high‐MW SLP shows weak homology to N‐acetyl muramoyl‐l‐alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high‐MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high‐MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription–polymerase chain reaction (RT–PCR) analysis to be transcribed.
Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats
Authors: Gumulak‐Smith Juliann , Teachman Amy , Tu Anh‐Hue T. , Simecka Jerry W. , Lindsey J. Russell , Dybvig Kevin
Published: March 2003

Restriction and modification (R–M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase‐variable R–M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R–M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony‐forming units (cfu) lacked R–M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R–M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R–M activity of the mycoplasmal cell population. We propose that variations in the production of R–M activity and cell surface proteins are important for the survival of the mycoplasma within the host.
Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA‐I adhesin
Authors: Benz Inga , Schmidt M. Alexander
Published: March 2003

The diffuse adherence of Escherichia coli strain 2787 (O126:H27) is mediated by the autotransporter adhesin AIDA‐I (adhesin‐involved‐in‐diffuse‐adherence) encoded by the plasmid‐borne aidA gene. AIDA‐I exhibits an aberrant mobility in denaturing gel electrophoresis. Deletion of the open reading frame (ORF) A immediately upstream of aidA restores the predicted mobility of AIDA‐I, but the adhesin is no longer functional. This indicates that the mature AIDA‐I adhesin is post‐translationally modified and the modification is essential for adherence function. Labelling with digoxigenin hydrazide shows AIDA‐I to be glycosylated. Using carbohydrate composition analysis, AIDA‐I contains exclusively heptose residues (ratio heptose:AIDA‐I ≈19:1). The deduced amino acid sequence of the cytoplasmic open reading frame (ORF) A gene product shows homologies to heptosyltransferases. In addition, the modification was completely abolished in an ADP–glycero‐manno‐heptopyranose mutant. Our results provide direct evidence for glycosylation of the AIDA‐I adhesin by heptoses with the ORF A gene product as a specific (mono)heptosyltransferase generating the functional mature AIDA‐I adhesin. Consequently, the ORF A gene has been denoted ‘aah’ (autotransporter‐adhesin‐heptosyltransferase). Glycosylation by heptoses represents a novel protein modification in eubacteria.
The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison–antidote system
Authors: Afif Hassan , Allali Noureddine , Couturier Martine , Van Melderen Laurence
Published: March 2003

The ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity. Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear. The results presented here indicate that, although CcdA is the DNA‐binding element of the CcdA–CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator–promoter region. Using electrophoretic mobility shift assays, we show that a (CcdA)2–(CcdB)2 complex binds DNA. The addition of extra CcdB to that protein–DNA complex completely abolishes DNA retardation. Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon. When the level of CcdA is superior or equal to that of CcdB, repression results. In contrast, derepression occurs when CcdB is in excess of CcdA. By ensuring an antidote–toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid‐containing bacteria.
The negative regulator Opi1 of phospholipid biosynthesis in yeast contacts the pleiotropic repressor Sin3 and the transcriptional activator Ino2
Authors: Wagner Christian , Dietz Martin , Wittmann Jürgen , Albrecht Antje , Schüller Hans‐Joachim
Published: March 2003

Structural genes of phospholipid biosynthesis in the yeastSaccharomyces cerevisiaeare transcriptionally co‐regulated by ICRE (inositol/choline‐responsive element) promoter motifs. Gene activation by an ICRE is mediated by binding of the Ino2/Ino4 transcription factor, whereas repression in the presence of high concentrations of inositol and choline (IC) requires an intact Opi1 repressor. However, the mechanism of specific repression and the functional interplay among these regulators remained unclear from previous work. Usingin vivoas well asin vitrointeraction assays, we show binding of the pleiotropic repressor Sin3 to the pathway‐specific regulator Opi1. The paired amphipathic helix 1 (PAH1) within Sin3 and OSID (Opi1–Sin3 interaction domain) in the N‐terminus of Opi1 were mapped as contact sites. The regulatory significance of the Opi1–Sin3 interaction was shown by the obvious deregulation of an ICRE‐dependent reporter gene in asin3mutant. Opi1 also interacts with a newly identified functional domain of the transcriptional activator Ino2 (RID, repressor interaction domain). These results define the molecular composition of the transcription complex mediating control of ICRE‐dependent genes and allow a hypothesis on the flow of regulatory information in response to phospholipid precursors.
PriA mutations that affect PriA–PriC function during replication restart
Authors: Sandler Steven J. , McCool Jesse D. , Do Tu Thien , Johansen Runa Ulsoe
Published: March 2003

In Escherichia coli, repair and restart of collapsed replication forks is thought to be essential for cell growth. The replication restart proteins, PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT and Rep, form redundant pathways that recognize repaired replication forks and restart them. Recognition, modulation of specific DNA structures and loading of the replicative helicase by the replication restart proteins, is likely to be important for replication restart. It has been hypothesized that PriB and PriC function with PriA in genetically separate and redundant PriA–PriB and PriA–PriC pathways. In this study, the del(priB)302 or priC303::kan mutations were used to isolate the PriA–PriB and PriA–PriC pathways genetically so that the effects of three priA missense mutations, priA300 (K230R), priA301 (C479Y) and priA306 (L557P), on these pathways could be assessed. In a wild‐type background, the three priA mutations had little, if any, effect on the phenotypes of UV resistance, basal levels of SOS expression and cell viability. In the priB mutant, priA300 and priA301 caused dramatic negative changes in the three phenotypes listed above (and others), whereas the third priA mutant allele, priA306, showed very little negative effect. In the priC mutant, all three priA mutations behaved similarly, producing little, if any, changes in phenotypes. We conclude that priA300 and priA301 mostly affect the PriA–PriC pathway and do so more than priA306. We suggest that PriA's helicase activity is important for the PriA–PriC pathway of replication restart.
KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake
Authors: Gage Matthew J. , Bruenn Jeremy , Fischer Marc , Sanders Dale , Smith Thomas J.
Published: March 2003

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies
, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage‐gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non‐specifically by high ionic strength buffers. We also demonstrate that KP4 affects 45Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calcium‐regulated signal transduction pathways.
A new family of aspartyl phosphate phosphatases targeting the sporulation transcription factor Spo0A of Bacillus subtilis
Authors: Perego Marta
Published: March 2003

The initiation of the sporulation developmental pathway in Bacillus subtilis is controlled by the phospho‐relay, a multicomponent signal transduction system. Multiple positive and negative signals are integrated by the phosphorelay through the opposing activities of histidine protein kinases and aspartyl phosphate phosphatases. Three members of the Rap family of phosphatases (RapA, RapB and RapE) specifically dephosphorylate the Spo0F∼P response regulator intermediate, while the Spo0A∼P transcription factor is specifically dephosphorylated by the Spo0E phosphatase and, as shown here, the newly identified YnzD and YisI proteins. The products of the YnzD and YisI genes are highly homologous to Spo0E and define a new family of phosphatases with a distinct signature motif in their amino acid sequence. As negative regulators of the developmental pathway, YnzD and YisI inhibit spore formation if over‐expressed, while a chromosomal deletion of their coding sequences results in increased sporulation frequency. Transcription of the ynzD, yisI and spo0E genes is differentially regulated and generally induced by growth conditions antithetical to sporulation. Negative signals interpreted by aspartyl phosphate phosphatases appear to be a common mechanism in Gram‐positive spore‐forming microorganisms.
Icm/Dot‐dependent upregulation of phagocytosis by Legionella pneumophila
Authors: Hilbi Hubert , Segal Gil , Shuman Howard A.
Published: March 2003

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild‐type L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild‐type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL‐60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid. Using fluorescence microscopy and green fluorescent protein (GFP)‐expressing strains, 10–20 times fewer icm/dot mutant bacteria were found to be internalized by HL‐60 cells and human monocyte‐derived macrophages (HMMΦ). Compared with icm/dot mutants, wild‐type L. pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15–70 bacteria per macrophage) that was not observed with icm/dot mutant strains. Wild‐type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMΦ. In addition, wild‐type L. pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s). Wild‐type L. pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co‐infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.
Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high‐frequency phase variation
Authors: Waite Richard D. , Struthers J. Keith , Dowson Christopher G.
Published: March 2003

The molecular genetic basis of high‐frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 34°C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11–239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high‐frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild‐type cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the ‘preprogrammed’ contingency genes in many Gram‐negative organisms that possess homopolymeric sequence repeats or motifs for site‐specific recombination.
Sister chromosome cohesion of Escherichia coli
Authors: Sunako Yumi , Onogi Toshinari , Hiraga Sota
Published: March 2003

We analysed Escherichia coli cells synchronized for initiation of chromosomal DNA replication by fluorescence in situ hybridization (FISH) using fluorescent DNA probes corresponding to various chromosomal regions. Sister copies of regions in an approximately oriC‐proximal half of the chromosome are cohesive with each other after replication until the late period of chromosome replication. Sister copies of regions relatively close to the terminus are also separated from each other in the same late period of replication. It is important that sister copies in all the tested regions are thus separated from each other nearly all at once in the late period of chromosome replication. These results are consistent with results obtained by FISH in randomly growing cultures. Cohesion of sister copies in an oriC‐close region is observed in a dam null mutant lacking DNA adenine methyltransferase the same as in the parental isogenic dam+ strain, indicating that the cohesion is independent of DNA adenine methyltransferase. This further implies that hemimethylated DNA‐binding proteins, such as SeqA, are not involved in the cohesion. On the other hand, the cohesion of sister copies of the oriC‐close region was not observed in mukB null mutant cells, suggesting that MukB might be involved in the chromosome cohesion.
Isolation and characterization of topological specificity mutants of minD in Bacillus subtilis
Authors: Karoui M. El , Errington Jeffery
Published: March 2003

In rod‐shaped bacteria such as Bacillus subtilis, division site selection is mediated by MinC and MinD, which together function as a division inhibitor. Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at mid‐cell. MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles. To investigate MinD activities further, we have constructed and analysed a collection of minD mutants. Mutations in the conserved ATPase motifs lead to an inactive protein, possibly unable to oligomerize, but which nevertheless retains some affinity for the cell membrane. Several mutations affecting the mid‐ to C‐terminal parts of MinD led to a protein probably unable to interact with DivIVA, but that could still stimulate division inhibition by MinC. These findings suggest that the ATPase activity of MinD is necessary for all its functions (possibly in part by controlling the oligomerization state of the protein). The other mutations may identify a surface of MinD involved in its interactions with DivIVA and a possible mechanism for control of MinD by DivIVA.
Formation of D‐alanyl‐lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes
Authors: Abachin Eric , Poyart Claire , Pellegrini Elisabeth , Milohanic Eliane , Fiedler Franz , Berche Patrick , Trieu‐Cuot Patrick
Published: March 2003

The dlt operon of Gram‐positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D‐alanine residues into the cell wall‐associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D‐Ala‐deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA− mutant did not show any morphological alterations and its growth rate was similar to that of the wild‐type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA− mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow‐derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA− mutant to non‐phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D‐alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.
Post‐transcriptional enhancement of Escherichia coli bgl operon silencing by limitation of BglG‐mediated antitermination at low transcription rates
Authors: Dole Sudhanshu , Kühn Sandra , Schnetz Karin
Published: March 2003

The silent bgl operon of Escherichia coli is activated by spontaneous
mutations that derepress its promoter. In addition, expression depends on specific
transcriptional antitermination within the operon by the antiterminator protein BglG.
Here, we show that BglG‐mediated antitermination limits expression of the bgl
operon when the cellular transcription rate is low. The expression levels of chromosomally
encoded activated bgl operon alleles are low but increase significantly when
BglG protein is provided in trans or when the expression is rendered inde‐pendent
of BglG‐mediated antitermination by mutation of the terminator. Plasmid‐encoded activated
bgl operon alleles are expressed at high levels. Moreover, a moderate (threefold)
further increase in the transcription rate of chromosomally encoded activated bgl
operon alleles in an rpoS mutant can result in high (up to 50‐fold increased)
expression levels. These data show that the expression of the bgl operon does
not correlate linearly with its cellular transcription rate. Moderate differences
in the transcription initiation rate are amplified post‐transcriptionally into large
changes in the expression level of the operon by the requirement of a threshold for
BglG‐mediated antitermination. Implications for bgl operon regulation by global regulators H‐NS, RpoS and others are discussed.
Functional analysis of the enteropathogenicEscherichia colitype III secretion system chaperone CesT identifies domains that mediate substrate interactions
Authors: Delahay Robin M. , Shaw Robert K. , Elliott Simon J. , Kaper James B. , Knutton Stuart , Frankel Gad
Published: March 2003

In many Gram‐negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion‐competent state. One such protein, CesT, serves a chaperone function for the enteropathogenicEscherichia coli(EPEC)translocatedintiminreceptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone–chaperone and chaperone–substrate associations, were determined. The CesT N‐terminal is implicated in chaperone dimerization, whereas the amphipathicα‐helical region of the C‐terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using theSalmonellaSicA chaperone to generate chaperone chimeras, we show that CesT–Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.
Identification of a gene (lpt‐3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis
Authors: Mackinnon Fiona G. , Cox Andrew D. , Plested Joyce S. , Tang Christoph M. , Makepeace Katherine , Coull Philip A. , Wright J. Claire , Chalmers Ronald , Hood Derek W. , Richards James C. , Moxon E. Richard
Published: March 2003

We have identified a gene, lpt‐3, that is required for the addition of phosphoethanolamine to the 3‐position (PEtn‐3) on the β‐chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn‐3 substituent is characteristic of the LPS of a majority (≈ 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface‐accessible epitope. All strains of Nm that have PEtn‐3 possess the lpt‐3 gene. In some lpt‐3‐containing strains, the 3‐position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt‐3 resulted in loss of PEtn‐3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt‐3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS‐based vaccine to protect against invasive meningococcal disease.
Protein phosphatase 2A on track for nutrient‐induced signalling in yeast
Authors: Zabrocki Piotr , Hoof Christine Van , Goris Jozef , Thevelein Johan M. , Winderickx Joris , Wera Stefaan
Published: March 2003

Early studies identified two bona fide protein phosphatase 2A (PP2A)‐encoding genes in Saccharo‐myces cerevisiae, designated PPH21 and PPH22. In

addition, three PP2A‐related phosphatases, encoded by PPH3, SIT4 and PPG1, have been

identified. All share as much as 86% sequence similarity at the amino acid

level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4

regulation will also be discussed. Whereas a role for PP2A in yeast morphology and

cell cycle has been readily recognized, uncovering its function in yeast signal transduction

is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A

and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP–PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient‐induced signalling in baker’s yeast.
Saccharomyces cerevisiae Ccr4–Not complex contributes to the control of Msn2p‐dependent transcription by the Ras/cAMP pathway
Authors: Lenssen E. , Oberholzer U. , Labarre J. , De Virgilio C. , Collart M. A.
Published: March 2003

The Ccr4–Not complex is a global regulator of transcription that affects genes positively and negatively and is thought to modulate the activity of TFIID. In the present work, we provide evidence that the Ccr4–Not complex may contribute to transcriptional regulation by the Ras/cAMP pathway. Several observations support this model. First, Msn2/4p‐dependent transcription, which is known to be under negative control of cAMP‐dependent protein kinase (PKA), is derepressed in all ccr4–not mutants. This phenotype is paralleled by specific post‐translational modification defects of Msn2p in ccr4–not mutants relative to wild‐type cells. Secondly, mutations in various NOT genes result in a synthetic temperature‐sensitive growth defect when combined with mutations that compromise cells for PKA activity and at least partially suppress the effects of both a dominant‐active RAS2Val‐19 allele and loss of Rim15p. Thirdly, Not3p and Not5p, which are modified and subsequently degraded by stress signals that also lead to increased Msn2/4p‐dependent activity, show a specific two‐hybrid interaction with Tpk2p. Together, our results suggest that the Ccr4–Not complex may function as an effector of the Ras/cAMP pathway that contributes to repress basal, stress‐ and starvation‐induced transcription by Msn2/4p.
Down‐regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein
Authors: Deghmane Ala‐Eddine , Giorgini Dario , Larribe Mireille , Alonso Jean‐Michel , Taha Muhamed‐Kheir
Published: March 2003

The initial attachment of Neisseria meningitidis to the target cell surface

appears to be largely pilus depend‐ent in capsulated bacteria. Intimate adhesion

subsequently occurs to permit colonization. We recently reported that insertional

inactivation of the crgA gene, which encodes a transcriptional regulator belonging

to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished

intimate adhesion. In this report, we analyse expression of the pilE and sia

genes, which are involved in the biosynthesis of pili and capsule respectively, during

bacteria–host cell interactions. Western blotting, transcriptional fusion

and reverse transcriptase polymerase chain reaction (RT‐PCR) analysis showed that

the expression of these genes was downregulated during intimate adhesion. DNA‐binding

assays, footprinting and RT‐PCR analysis indicated that this downregulation was directly

mediated by the CrgA protein. The pilE and sia promoters were found

to have a CrgA binding motif in common. These results strongly suggest that N.

meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.
Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection
Authors: Smith Alexander W. , Roche Hazeline , Trombe Marie‐Claude , Briles David E. , Håkansson Anders
Published: March 2003

In the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Strepto‐coccus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in α‐galactoside meta‐bolism and galactose transport. DLDH is an enzyme that is classically involved in the three‐step conversion of 2‐oxo acids to their respective acyl‐CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N‐terminal lipoyl domain. DLDH‐negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. These DLDH‐negative bacteria grew normally in vitro but were avirulent in sepsis and lung infection models in mice, indicating that DLDH activity is necessary for the survival of pneumococci within the host. The lack of virulence was not associated with a loss of 2‐oxo acid dehydrogenase activity, as the wild‐type pneumococcal strains did not contain activity of any of the known 2‐oxo acid enzyme complexes. Instead, studies of carbohydrate utilization demonstrated that the DLDH‐negative bacteria were impaired for α‐galactoside and galactose metabolism. The DLDH mutants lost their ability to oxidize or grow with galactose or melibiose as sole carbon source and showed reduced oxidation and growth on raffinose or stachyose. The bacteria had an 85% reduction in α‐galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH‐negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism.
PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding
Authors: Gómez Dennis , Cubero Beatriz , Cecchetto Gianna , Scazzocchio Claudio
Published: March 2003

The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG‐N‐CCGG inverted repeats and to CCGG‐6/7N‐CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG‐6N‐CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG‐N‐CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino‐terminal to it. Three high‐affinity sites in two different promoters have been visualized by in vivo methy‐lation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high‐affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiæ protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.
Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit‐specific strains of enteropathogenic Escherichia coli
Authors: Tauschek Marija , Strugnell Richard A. , Robins‐Browne Roy M.
Published: March 2003

We have characterized the LEE pathogenicity islands (PAIs) of two rabbit‐specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H‐) and 84/110‐1
(O103:H2), and have compared them to homologous loci from the human enteropathogenic
and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC
strain, RDEC‐1. All five PAIs contain a 34 kb core region that is highly conserved
in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly
larger (59 540 basepairs) than those of the human strains, which are less than
44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb
core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA
and senA. The LEE of 84/110‐1 is approximately 85 kb and is located at pheV
tRNA. Its core is almost identical to those of 83/39 and RDEC‐1, apart from a larger
espF gene, but its flanking regions contain trcA, a putative virulence
determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int‐phe.
The LEE PAI of 84/110‐1 is also flanked by short direct repeats (representing the
3′‐end of pheV tRNA), suggesting that it may be unstable. To
investigate this possibility, we constructed a LEE::sacB derivative of 84/110‐1
and showed that the PAI was capable of spontaneous deletion. We also showed that
Int‐phe can mediate site‐specific integration of foreign DNA at the pheU tRNA
locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.
The Escherichia coli FKBP‐type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage φX174
Authors: Bernhardt Thomas G. , Roof William D. , Young Ry
Published: March 2003

Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage φX174 uses a single lysis gene, E, encoding a 91‐amino‐acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E‐mediated lysis and φX174 plaque formation. The slyD gene encodes a FKBP‐type peptidyl‐prolyl cis‐trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP‐dependence, spontaneous plaque‐forming mutants of φX174 were isolated on a slyD lawn. All of these Epos (‘plates on slyD’) suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c‐myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse‐chase labelling ex‐periments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis‐sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD‐ dependence phenotype for identifying elements of PPIase selectivity.
Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice
Authors: Bai Chen , Ramanan Narendrakumar , Wang Yan Ming , Wang Yue
Published: March 2003

Here, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SAC‐defective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild‐type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti‐C. albicans therapies.
The ramC gene is required for morphogenesis in Streptomyces coelicolor and expressed in a cell type‐specific manner under the direct control of RamR
Authors: O'Connor Tamara J. , Kanellis Pamela , Nodwell Justin R.
Published: March 2003

The bacterium Streptomyces coelicolor produces two cell types during the course of its life cycle: the aerial hyphae, which metamorphose into spores, and the substrate hyphae, which synthesize antibiotics. We show that the genes ramC and ramR are required for the production of the aerial hyphae but are dispensable for vegetative growth and antibiotic synthesis. We find that ramC is expressed in the substrate hyphae and shut off in the aerial hyphae by the time visible signs of sporulation‐associated septation are evident. Production of RamC requires the developmental regulators bldD, cprA and ramR, but not bldM or bldN, and we show that the RamR protein interacts directly with DNA in the ramC promoter region sug‐gesting that it is, at least in part, responsible for regulating ramC expression.
LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli
Authors: Lehnen D. , Blumer C. , Polen T. , Wackwitz B. , Wendisch V. F. , Unden G.
Published: March 2003

The function of the LysR‐type regulator LrhA of Escherichia coli was defined by comparing whole‐genome mRNA profiles from wild‐type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent KD≈ 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD2C2) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD2C2 is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD2C2.
Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35‐amino‐acid cell‐killing peptide and a cis‐encoded small antisense RNA in Escherichia coli
Authors: Kawano Mitsuoki , Oshima Taku , Kasai Hiroaki , Mori Hirotada
Published: March 2003

Genome sequence analyses of Escherichia coli K‐12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR‐A, ‐B, ‐C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR‐D), 450 bp in length and nearly identical to LDR‐A, ‐B and ‐C, is located at 79.7 min, a position that is directly opposite the position of LDR‐A, ‐B and ‐C. In this study, we demonstrate that LDR‐D encodes a 35‐amino‐acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (≈ 370 nucleotides) encoding LdrD and an unstable cis‐encoded antisense RNA (≈ 60 nucleotides), which functions as a trans‐acting regulator of ldrD translation. We propose that LDR encodes a toxin–antitoxin module. LDR‐homologous sequences are not present on any known plasmids but are conserved in Salmonella and other enterobacterial species.
Imp/OstA is required for cell envelope biogenesis in Escherichia coli
Authors: Braun Martin , Silhavy Thomas J.
Published: March 2003

In Gram‐negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high‐molecular‐weight disulphide‐bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane.
CPMK2, an SLT2‐homologous mitogen‐activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis‐related MAP kinase cascade in phytopathogenic fungi
Authors: Mey Géraldine , Held Katrin , Scheffer Jan , Tenberge Klaus B. , Tudzynski Paul
Published: March 2003

Cpmk2
, encoding a mitogen‐activated protein (MAP) kinase from the ascomycete
Claviceps purpurea
, is an orthologue of
SLT2
from
Saccharomyces cerevisiae
, the first isolated from a biotrophic, non‐appressorium‐forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen,
Magnaporthe grisea
, that have been deprived of their MAP kinase gene
mps1
, the ability of
cpmk2
to complement the defects of Δ
mps1
was investigated. Interestingly, the
C. purpurea
gene, under the control of its own promoter, was able to complement the
M. grisea
mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2
/ MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.
Type III‐dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell
Authors: Szurek Boris , Rossier Ombeline , Hause Gerd , Bonas Ulla
Published: March 2003

Many plant pathogenic bacteria utilize a conserved type III secretion system (TTSS) to deliver effector proteins into the host tissue. Indirect evidence has suggested that at least some effector proteins are translocated from the bacterial cytoplasm into the plant cell. Using an immunocytochemical approach, we demonstrate that the type III effector AvrBs3 from Xanthomonas campestris pv. vesicatoria localizes to nuclei of infected pepper leaves. Importantly, AvrBs3 translocation was observed in situ in native tissues of susceptible and resistant plants. AvrBs3 was detected in the nucleus as soon as 4 h post infection, which was dependent on a functional TTSS and the putative translocator HrpF. N‐terminal AvrBs3 deletion derivatives are no longer secreted by the TTSS in vitro and could not be detected inside the host cells, suggesting that the N‐terminus of AvrBs3 is important for secretion. Deletion of the nuclear localization signals in the AvrBs3 C‐terminus, which are required for the AvrBs3‐mediated induction of the hypersensitive reaction in resistant pepper plants, abolished AvrBs3 localization to the nucleus. This is the first report on direct evidence for translocation of a native type III effector protein from a plant pathogenic bacterium into the host cell.