bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory and functional co‐operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease
Authors: Barnich Nicolas , Boudeau Jérôme , Claret Laurent , Darfeuille‐Michaud Arlette 
Published: December 2004

SummaryGenetic determinants that co‐operate with type 1 pili to mediate invasion were sought for in adherent‐invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82‐ΔfliC deleted for the flagellin‐encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off‐position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82‐ΔfliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co‐ordinate expression of invasive determinants.
Replication restart in gyrB Escherichia coli mutants
Authors: Grompone Gianfranco , Ehrlich S. Dusko , Michel Bénédicte 
Published: December 2004

SummaryGyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co‐lethal in the presence of a ΔtopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA‐dependent primosome assembly, directly on the in‐activated replication forks, without the formation of a recombination intermediate.
Dicarboxylic amino acids and glycine‐betaine regulate chaperone‐mediated protein‐disaggregation under stress
Authors: Diamant Sophia , Rosenthal David , Azem Abdussalam , Eliahu Noa , Ben‐Zvi Anat Peres , Goloubinoff Pierre 
Published: December 2004

SummaryActive protein‐disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress‐induced protein aggregates in vitro and in Escherichia coli cells. K‐glutamate and glycine‐betaine (betaine) naturally accumulate in salt‐stressed cells. In addition to providing thermo‐protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone‐mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K‐glutamate or betaine. The combined protective, counter‐negative and net activatory effects of K‐glutamate and betaine, allowed protein disaggregation and refolding under heat‐shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte‐activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat‐shock conditions.
The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology
Authors: Hume Peter J. , McGhie Emma J. , Hayward Richard D. , Koronakis Vassilis 
Published: December 2004

SummaryAn essential early event in Shigella and Salmonella pathogenesis is invasion of non‐phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full‐length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N‐terminal domain predicted to form a trimeric coiled‐coil, and is predominantly α‐helical. Upon lipid interaction, two transmembrane domains (residues 313–333 and 399–419) penetrate the bilayer, allowing the intervening hydrophilic region (334–398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB‐derived 162 residue α‐helical polypeptide (IpaB418−580) is a potent inhibitor of IpaB‐directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10−8 M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB418−580 failed to prevent the contact‐dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact‐dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.
Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast
Authors: El Alami Mohamed , Messenguy Francine , Scherens Bart , Dubois Evelyne 
Published: December 2004

SummaryIn Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82Δ or kcs1Δ cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR‐regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine‐responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1‐dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS‐box proteins against degradation.
A MAP kinase cascade composed of cell type specific and non‐specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans
Authors: Davidson Robert C. , Nichols Connie B. , Cox Gary M. , Perfect John R. , Heitman Joseph 
Published: December 2004

Summary Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the α allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating‐type specific (Ste11α, Ste12α) and non‐specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type‐specific differentiation but not for virulence. The Ste11α, Ste7 and Cpk1 kinases were found to act as a co‐ordinate signalling module, whereas the Ste12α transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type‐specific and non‐specific components, yielding pathways that contribute to cell type‐specific patterns of signalling and differentiation.
Ty3 requires yeast La homologous protein for wild‐type frequencies of transposition
Authors: Aye Michael , Sandmeyer Suzanne B. 
Published: December 2004

SummaryThe Saccharomyces cerevisiae retrovirus‐like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild‐type levels, assembled into virus‐like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild‐type control at early times, but approached the wild‐type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid‐borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target‐specific step, such as integration.
Downregulation of the heat shock response is independent of DnaK and σ32 levels in Caulobacter crescentus
Authors: Da Silva Antonio C.A. , Simão Rita C.G. , Susin Michelle F. , Baldini Regina L. , Avedissian Marcelo , Gomes Suely L. 
Published: December 2004

SummaryExpression of heat shock genes in Gram‐negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the σ32 subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though σ32 levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of σ32 and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on σ32 levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between σ32 and σ73, the major sigma factor, for the core RNAP could be the most important factor controlling the shut‐off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of σ73 gene is heat shock inducible.
Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance
Authors: Land Kirkwood M. , Delgadillo‐Correa Maria G. , Tachezy Jan , Vanacova Stepanka , Hsieh Christine L. , Sutak Robert , Johnson Patricia J. 
Published: December 2004

SummaryFerredoxin, Fd, is often deficient in metronidazole‐resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by ∼2.6 and ∼2.0 kBp of the Fd 5′ and 3′ flanking regions (pKO‐FD‐NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO‐FD‐NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.
Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis
Authors: Bunn Michael W. , Ordal George W. 
Published: December 2004

SummaryAddition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C‐terminal domain. We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141−357)). Thus, in undergoing the transition from unbound receptor to ligand‐bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation. The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced. However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced. Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141−357) was present. Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C‐terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins.
Lack of O‐antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica
Authors: Kukkonen Maini , Suomalainen Marjo , Kyllönen Päivi , Lähteenmäki Kaarina , Lång Hannu , Virkola Ritva , Helander Ilkka M. , Holst Otto , Korhonen Timo K. 
Published: December 2004

SummaryThe O‐antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β‐barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O‐antigen repeats on wild‐type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O‐antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6‐Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla‐mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O‐antigen prevented PgtE‐mediated bacterial adhesion to basement membrane. Substitution of Arg‐138 and Arg‐171 of the motif for protein binding to lipid A 4′‐phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O‐antigen sterically prevents recognition of large‐molecular‐weight substrates. Loss of O‐antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.
Genetics of capsule O‐acetylation in serogroup C, W‐135 and Y meningococci
Authors: Claus Heike , Borrow Ray , Achtman Mark , Morelli Giovanna , Kantelberg Carmen , Longworth Emma , Frosch Matthias , Vogel Ulrich 
Published: December 2004

SummaryCapsular polysaccharides of serogroup C, W‐135 and Y meningococci were previously reported to be O‐acetylated at the sialic acid residues. There is evidence that O‐acetylation affects the immunogenicity of polysaccharide vaccines. We identified genes indispensable for O‐acetylation of serogroup C, W‐135 and Y meningococci downstream of the capsule synthesis genes siaA–D. The genes were co‐transcribed with the sia operon as shown by reverse transcription polymerase chain reaction analysis. The putative capsular polysaccharide O‐acetyltransferases were designated OatC and OatWY. The protein OatWY of serogroups W‐135 and Y showed sequence homologies to members of the NodL–LacA–CysE family of bacterial acetyltransferases, whereas no sequence homology with any known protein in the different databases was found for the serogroup C protein OatC. In serogroup W‐135 and Y meningococci, several clonal lineages either lacked OatWY or OatWY was inactivated by insertion of IS1301. For serogroup C meningococci, we observed in vitro phase variation of O‐acetylation, which resulted from slipped‐strand mispairing in homopolymeric tracts. This finding explains the observation of naturally occurring de‐O‐acetylated serogroup C meningococci. Our report is the first description of sequences of sialic acid O‐acetyltransferase genes that have not been cloned from either other bacterial or mammalian organisms.
Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum
Authors: Otto Grant P. , Wu Mary Y. , Clarke Margaret , Lu Hao , Anderson O. Roger , Hilbi Hubert , Shuman Howard A. , Kessin Richard H. 
Published: December 2004

SummaryThe Gram‐negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free‐living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy‐inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild‐type D. discoideum. Furthermore, a green fluorescent protein (GFP)‐tagged marker of autophagosomes, Apg8, does not systematically co‐localize with DsRed‐labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.
Cross‐species hybridization of a Borrelia burgdorferi DNA array reveals infection‐ and culture‐associated genes of the unsequenced genome of the relapsing fever agent Borrelia hermsii
Authors: Zhong Jianmin , Barbour Alan G. 
Published: December 2004

SummaryThe known genome sequence of Borrelia burgdorferi, an agent of Lyme borreliosis, was used to study the genetic content and gene expression in B. hermsii, another spirochete pathogen and a cause of relapsing fever. Cross‐species hybridization of a DNA array representing 1628 open reading frames (ORF) of B. burgdorferi with genomic DNA of B. hermsii indicated that the latter organism has at least 81% of the chromosomal genes and 43% of the plasmid genes of B. burgdorferi. We then carried out quantitative hybridization of the arrays with multiple replicates of cDNA produced from B. hermsii cells growing in the blood of infected mice or in culture medium that was adjusted to the same pH, temperature and a spirochete density as infected blood. Of 642 B. burgdorferi ORFs hybridized by all replicates under both conditions, 12 (1.9%) demonstrated differential expression by a regularized t‐test and stringent criteria. BBP07 and BBG30, two plasmid‐borne ORFs with the greatest measurable difference in expression between in vivo and in vitro conditions, putatively encode proteins of unknown function. Orthologues of BBP07 in B. hermsii were identified, and increased expression in infected mice was demonstrated by quantitative reverse‐transcriptase polymerase chain reaction.
Several distinct localization patterns for penicillin‐binding proteins in Bacillus subtilis
Authors: Scheffers Dirk‐Jan , Jones Laura J. F. , Errington Jeffery 
Published: December 2004

SummaryBacterial cell shape is determined by a rigid external cell wall. In most non‐coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin‐binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild‐type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP–PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.