bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence
Authors: Frees Dorte , Qazi Saara N. A. , Hill Philip J. , Ingmer Hanne 
Published: June 2003

SummaryClp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature‐tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325‐4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall‐associated proteins. In the mutant cells, the amount of α‐haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum‐sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.
Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants
Authors: Klausen Mikkel , Heydorn Arne , Ragas Paula , Lambertsen Lotte , Aaes‐Jørgensen Anders , Molin Søren , Tolker‐Nielsen Tim 
Published: June 2003

SummaryBiofilm formation by Gfp‐tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time‐lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild‐type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild‐type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild‐type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.
Autoinduction and signal transduction in the regulation of staphylococcal virulence
Authors: Novick Richard P. 
Published: June 2003

SummaryThe accessory genes of Staphylococcus aureus, in‐cluding those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two‐component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time‐ and population density‐dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full‐scale test.
Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori
Authors: Schirm M. , Soo E. C. , Aubry A. J. , Austin J. , Thibault P. , Logan S. M. 
Published: June 2003

SummaryMass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post‐translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7‐diacetamido‐3,5,7,9‐tetradeoxy‐l‐glycero‐l‐manno ‐n o n‐ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non‐motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis‐electrospray mass spectrometry (CE‐ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide‐activated form of Pse, CMP‐Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono‐ and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.
Identification of nuclear proteins that interact differentially with Plasmodium falciparum var gene promoters
Authors: Voss Till S. , Kaestli Mirjam , Vogel Denise , Bopp Selina , Beck Hans‐Peter 
Published: June 2003

SummaryThe Plasmodium falciparum virulence factor PfEMP1 is responsible for both antigenic variation and cytoadherence of infected erythrocytes in malaria. Approximately 50 var genes per parasite genome code for this highly polymorphic surface protein. We showed recently that chromosome‐central and subtelomeric var genes are controlled by different promoters. Here, we report that transcriptional repression of var genes located in different chromosomal regions occurs by different mechanisms. Subtelomeric var gene transcription is repressed 4–8 h before that of chromosome‐central var genes. Both repression events coincide with the shifted expression of two distinct nuclear proteins binding specifically to conserved sequence motifs, SPE1 and CPE, present in the respective promoter. Furthermore, a reiterated and highly conserved subtelomeric var promoter element (SPE2) interacts with a nuclear factor exclusively expressed during S‐phase. Promoter analysis by transient transfection suggested direct involvement of these interactions in var gene repression and silencing, and identified regions implicated in transcriptional activation of var genes.
Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein
Authors: Rosenberg Emiko Y. , Bertenthal Dan , Nilles Matthew L. , Bertrand Kevin P. , Nikaido Hiroshi 
Published: June 2003

SummaryAcrAB of Escherichia coli, an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low‐molecular‐weight inducers specifically bind to the C‐terminal, non‐DNA‐binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre‐existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407–1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli, were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob‐mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.
Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes
Authors: Machner Matthias P. , Frese Susanne , Schubert Wolf‐Dieter , Orian‐Rousseau Veronique , Gherardi Ermanno , Wehland Jürgen , Niemann Hartmut H. , Heinz Dirk W. 
Published: June 2003

SummaryThe surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine‐rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface‐exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB′, a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full‐length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.
Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes
Authors: Hansen Anne‐Marie , Lehnherr Hansjörg , Wang Xiandong , Mobley Victoria , Jin Ding Jun 
Published: June 2003

SummaryThe stringent starvation protein A (SspA), an Escherichia coli RNA polymerase (RNAP)‐associated protein, has been reported to be essential for lytic growth of bacteriophage P1. Unlike P1 early promoters, P1 late promoters are not recognized by RNAP alone. A phage‐encoded early protein, Lpa (late promoter activator protein, formerly called gp10), has been shown to be required for P1 late transcription in vivo. Here, we demonstrate that SspA is a transcription activator for P1 late genes. Our results indicated that Lpa is not limiting in an sspA mutant. However, the transcription of P1 late genes was deficient in an sspA mutant in vivo. We demonstrated that SspA/Lpa are required for transcription activation of the P1 late promoter Ps in vitro. In addition, SspA and Lpa were shown to facilitate the binding of RNAP to Ps late promoter DNA. Activation of late transcription by SspA/Lpa was dependent on holoenzyme containing σ70 but not σS, indicating that the two activators discriminate between the two forms of the holoenzyme. Furthermore, P1 early gene expression was downregulated in the wild‐type background, whereas it persisted in the sspA mutant background, indicating that SspA/Lpa mediate the transcriptional switch from the early to the late genes during P1 lytic growth. Thus, this work provides the first evidence for a function of the E. coli RNAP‐associated protein SspA.
Isolation of Listeria monocytogenes mutants with high‐level in vitro expression of host cytosol‐induced gene products
Authors: Shetron‐Rama Lynne M. , Mueller Kimberly , Bravo Juan M. , Bouwer H. G. Archie , Way Sing Sing , Freitag Nancy E. 
Published: June 2003

SummaryThe facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell‐to‐cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200‐fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high‐level expression of PrfA‐dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild‐type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA‐dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.
Global regulation by CsrA in Salmonella typhimurium
Authors: Lawhon Sara D. , Frye Jonathan G. , Suyemoto Mitsu , Porwollik Steffen , McClelland Michael , Altier Craig 
Published: June 2003

SummaryCsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI‐1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI‐1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non‐motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2‐propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co‐ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2‐propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co‐ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.
Regulation of Vibrio vulnificus virulence by the LuxS quorum‐sensing system
Authors: Kim Soo Young , Lee Shee Eun , Kim Young Ran , Kim Choon Mee , Ryu Phil Youl , Choy Hyon E. , Chung Sun Sik , Rhee Joon Haeng 
Published: June 2003

Summary Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections. We tested whether V. vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum‐sensing system 1 and/or 2. Although there was no evidence for signalling system 1, we found that V. vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V. harveyi through signalling system 2. Maximal autoinducer 2 (AI‐2) activity was observed during mid‐exponential to early stationary phase and disappeared in the late stationary phase when V. vulnificus was grown in heart infusion broth containing 2.5% NaCl. V. vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0). From a cosmid library of V. vulnificus type strain ATCC 29307, we have identified the AI‐2 synthase gene (luxSVv) showing 80% identity with that of V. harveyi (luxSVh) at the amino acid level. To investigate the pathogenic role of luxSVv, a deletion mutant of the clinical isolate V. vulnificus MO6‐24/O was constructed. The luxSVv mutant showed a significant delay in protease production and an increase in haemolysin production. The decreased protease and increased haemolysin activities were restored to the isogenic wild‐type level by complementation with the wild‐type luxSVv allele. The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild‐type bacteria. Transcriptional activities of the haemolysin gene (vvhA) and protease gene (vvpE) were also observed in the mutant using chromosomal PvvhA::lacZ and PvvpE::lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation. The mutation resulted in an attenuation of lethality to mice. Intraperitoneal LD50 of the luxSVv mutant increased by 10‐ and 750‐fold in ferric ammonium citrate‐non‐overloaded and ferric ammonium citrate‐overloaded mice respectively. The time required for the death of mice was also significantly delayed in the luxSVv mutant. Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation. Taken together, the V. vulnificus LuxS quorum‐sensing system seems to play an important role in co‐ordinating the expression of virulence factors.
Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi
Authors: Alverson Janet , Bundle Sharyl F. , Sohaskey Charles D. , Lybecker Meghan C. , Samuels D. Scott 
Published: June 2003

SummaryOspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23°C compared with cultures grown at 35–37°C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A1, an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23°C to 35°C or after adding coumermycin A1. In addition, OspC synthesis was higher in a gyrB mutant than in wild‐type B. burgdorferi. Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi, but not in Escherichia coli, as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.
Ribosome–DnaK interactions in relation to protein folding
Authors: Ghosh Jaydip , Basu Arunima , Pal Saumen , Chowdhuri Saheli , Bhattacharya Arpita , Pal Debashis , Chattoraj Dhruba K. , DasGupta Chanchal 
Published: June 2003

SummaryBacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.
Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans
Authors: Semighini Camile P. , Von Zeska Kress Fagundes Márcia Regina , Ferreira Joseane Cristina , Pascon Renata Castiglioni , De Souza Goldman Maria Helena , Goldman Gustavo Henrique 
Published: June 2003

SummaryThe Mre11‐Rad50‐Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti‐topoisomerase I drug camptothecin. We have used the scaANBS1 cDNA as a bait in a yeast two‐hybrid screening and report the identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra‐S‐phase checkpoint. ScaANBS1 also participates in G2‐M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.