bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis
Authors: Srinivasa Rao P. S. , Yamada Yoshiyuki , Tan Yuen Peng , Leung Ka Yin 
Published: July 2004

Summary Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro‐ and extraintestinal infections in humans. Using a combination of comparative proteomics and TnphoA mutagenesis, we have identified five proteins that may contribute to E. tarda PPD130/91 pathogenesis. Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM). Five out of six proteins could be identified by their mass spectra. Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species. The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri. The complete sequencing and homology studies of evpA–H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions. Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami. Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E. tarda pathogenesis. The transport of the EvpC protein may not use the TTSS in E. tarda. The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37°C), and disruption of esrB affected their expression. The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E. tarda pathogenesis.
Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus
Authors: Leonard Thomas A. , Butler P. Jonathan G. , Löwe Jan 
Published: July 2004

SummaryProkaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The plasmid partitioning loci encode two proteins, ParA and ParB, and a cis‐acting centromere‐like site denoted parS. The chromosomally encoded homologues of ParA and ParB, Soj and Spo0J, play an active role in chromosome segregation during bacterial cell division and sporulation. Spo0J is a DNA‐binding protein that binds to parS sites in vivo. We have solved the X‐ray crystal structure of a C‐terminally truncated Spo0J (amino acids 1–222) from Thermus thermophilus to 2.3 Å resolution by multiwavelength anomalous dispersion. It is a DNA‐binding protein with structural similarity to the helix–turn–helix (HTH) motif of the lambda repressor DNA‐binding domain. The crystal structure is an antiparallel dimer with the recognition α‐helices of the HTH motifs of each monomer separated by a distance of 34 Å corresponding to the length of the helical repeat of B‐DNA. Sedimentation velocity and equilibrium ultracentrifugation studies show that full‐length Spo0J exists in a monomer–dimer equilibrium in solution and that Spo0J1–222 is exclusively monomeric. Sedimentation of the C‐terminal domain of Spo0J shows it to be exclusively dimeric, confirming that the C‐terminus is the primary dimerization domain. We hypothesize that the C‐terminus mediates dimerization of Spo0J, thereby effectively increasing the local concentration of the N‐termini, which most probably dimerize, as shown by our structure, upon binding to a cognate parS site.
Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched‐chain amino acids
Authors: Shivers Robert P. , Sonenshein Abraham L. 
Published: July 2004

SummaryCodY, a GTP‐activated global transcriptional regulator of early stationary phase genes, is conserved in many Gram‐positive bacterial species. Recently, a number of novel targets regulated by CodY have been identified, including three Bacillus subtilis operons involved in branched‐chain amino acid (BCAA) biosynthesis (Molle, V., et al., 2003, J Bacteriol 185: 1911–1922). The mechanism of involvement of CodY in regulating the ilvB operon was investigated here using in vivo transcriptional fusions, in vitro gel mobility shift assays and DNase I footprinting assays. CodY was found to mediate regulation of the ilvB operon by GTP and BCAAs and to bind to the ilvB promoter region. BCAAs increased the affinity of CodY for the ilvB promoter and for all other CodY targets tested. This effect of BCAAs in vitro was additive with the effect of GTP on CodY DNA‐binding activity.
Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori
Authors: Bury‐Moné Stéphanie , Thiberge Jean‐Michel , Contreras Monica , Maitournam Aboubakar , Labigne Agnès , De Reuse Hilde 
Published: July 2004

SummaryThe virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real‐time polymerase chain reaction, dot‐blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC‐HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal‐responsive transcriptional regulators (NikR and Fur) and an essential two‐component response regulator (HP0166, OmpR‐like) as effectors of the H. pylori acid response. Finally, a nikR‐fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.
FbpA, a novel multifunctional Listeria monocytogenes virulence factor
Authors: Dramsi S. , Bourdichon F. , Cabanes D. , Lecuit M. , Fsihi H. , Cossart P. 
Published: July 2004

Summary Listeria monocytogenes is a Gram‐positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature‐tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E‐cadherin. fbpA encodes a 570‐amino‐acid polypeptide that has strong homologies to atypical fibronectin‐binding proteins. FbpA binds to immobilized human fibronectin in a dose‐dependent and saturable manner and increases adherence of wild‐type L. monocytogenes to HEp‐2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co‐immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin‐binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.
Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence
Authors: Watson Michael E. , Jarisch Justin , Smith Arnold L. 
Published: July 2004

SummaryMutants in deoxyadenosine methyltransferase (dam) from many Gram‐negative pathogens suggest multiple roles for Dam methylase: directing post‐replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT‐1; restriction with Dam methylation‐sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2‐aminopurine‐susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI‐H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam‐regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT‐1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain‐dependent fashion and may function as a regulator of gene expression including virulence factors.
The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins
Authors: Claessen Dennis , Stokroos Ietse , Deelstra Heine J. , Penninga Nynke A. , Bormann Christiane , Salas José A. , Dijkhuizen Lubbert , Wösten Han A. B. 
Published: July 2004

SummaryStreptomycetes form hydrophobic aerial hyphae that eventually septate into hydrophobic spores. Both aerial hyphae and spores possess a typical surface layer called the rodlet layer. We present here evidence that rodlet formation is conserved in the streptomycetes. The formation of the rodlet layer is the result of the interplay between rodlins and chaplins. A strain of Streptomyces coelicolor in which the rodlin genes rdlA and/or rdlB were deleted no longer formed the rodlet layer. Instead, these surfaces were decorated with fine fibrils. Deletion of all eight chaplin genes (strain ΔchpABCDEFGH) resulted in the absence of the rodlet layer as well as the fibrils at surfaces of aerial hyphae and spores. Apart from coating these surfaces, chaplins are involved in the escape of hyphae into the air, as was shown by the strong reduction in the number of aerial hyphae in the ΔchpABCDEFGH strain. The decrease in the number of aerial hyphae correlated with a lower expression of the rdl genes in the colony. Yet, expression per aerial hypha was similar to that in the wild‐type strain, indicating that expression of the rdl genes is initiated after the hypha has sensed that it has grown into the air.
IS231–MIC231 elements from Bacillus cereus sensu lato are modular
Authors: De Palmenaer Daniel , Vermeiren Céline , Mahillon Jacques 
Published: July 2004

SummaryIS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene. A first major variation of this canonical organization was found in MIC231A1. This mobile insertion cassette (MIC), delineated by IS231A‐related extremities, contained an active d‐stereospecific endopeptidase (adp) gene instead of a transposase. Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase. In this paper, we show that this family of IS231–MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity. Several IS231A‐like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors. Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes. Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant. In vivo transposition assays showed that MIC231V is also trans‐activated by the IS231A transposase. These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B. thuringiensis group.
Activation of transcription initiation from a stable RNA promoter by a Fis protein‐mediated DNA structural transmission mechanism
Authors: Opel Michael L. , Aeling Kimberly A. , Holmes Walter M. , Johnson Reid C. , Benham Craig J. , Hatfield G. Wesley 
Published: July 2004

SummaryThe leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors. Transcription from this and other stable RNA promoters is known to be affected by a cis‐acting UP element and by Fis protein interactions with the carboxyl‐terminal domain of the α‐subunits of RNA polymerase. In this report, we suggest that transcription from the leuV promoter also is activated by a Fis‐mediated, DNA supercoiling‐dependent mechanism similar to the IHF‐mediated mechanism described previously for the ilvPG promoter (S. D. Sheridan et al., 1998, J Biol Chem 273: 21298–21308). We present evidence that Fis binding results in the translocation of superhelical energy from the promoter‐distal portion of a supercoiling‐induced DNA duplex destabilized (SIDD) region to the promoter‐proximal portion of the leuV promoter that is unwound within the open complex. A mutant Fis protein, which is defective in contacting the carboxyl‐terminal domain of the α‐subunits of RNA polymerase, remains competent for stimulating open complex formation, suggesting that this DNA supercoiling‐dependent component of Fis‐mediated activation occurs in the absence of specific protein interactions between Fis and RNA polymerase. Fis‐mediated translocation of superhelical energy from upstream binding sites to the promoter region may be a general feature of Fis‐mediated activation of transcription at stable RNA promoters, which often contain A+T‐rich upstream sequences.
Dissection of septin actin interactions using actin overexpression in Saccharomyces cerevisiae
Authors: Norden Caren , Liakopoulos Dimitris , Barral Yves 
Published: July 2004

SummaryAlthough many proteins can be overexpressed several fold without much effect on cell viability and morphology, some become toxic upon a slight increase in their intracellular level. This is particularly true for cytoskeletal proteins and has proven useful in the past for studying the cytoskeleton. In yeast, actin and tubulin are examples of proteins that cannot be overexpressed without affecting cell viability. Here, we have analysed the effect of actin overexpression in Saccharomyces cerevisiae. We show that actin overexpression interferes differently with distinct aspects of actin function. For example, two‐ to fourfold overexpression of actin did not affect the establishment of actin polarity, whereas it abrogated its maintenance. Also, actin structures that are barely visible in wild‐type cells could be observed upon actin overexpression. This allowed us to identify a new ring‐like actin structure genetically distinguishable from the actomyosin contractile ring. Formation of this actin structure upon actin overexpression was dependent on the septin cytoskeleton, the poorly understood cytokinetic protein Hof1 and the Arp2/3 complex. In contrast to the actomyosin ring, the ring formed upon actin overexpression required neither Myo1 nor formins for assembly. Therefore, we propose that Hof1 acts as a linker between actin and septins. Furthermore, we found that, in the absence of actin overexpression, a novel, Hof1‐dependent actin belt is formed at the bud neck of anaphase cells. The physiological role of this belt might be related to that of the similar structure observed in dividing fission yeast.
In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II‐Gly5) of Staphylococcus aureus
Authors: Schneider Tanja , Senn Maria Magdalena , Berger‐Bächi Brigitte , Tossi Alessandro , Sahl Hans‐Georg , Wiedemann Imke 
Published: July 2004

Summary Staphylococcus aureus peptidoglycan is cross‐linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t‐RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP‐MurNAc‐pentapeptide, lipid I and lipid II and the staphylococcal t‐RNA pool, as well as His‐tagged Gly‐tRNA‐synthetase and His‐tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II‐Gly1 and lipid II‐Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane‐associated steps of S. aureus peptidoglycan biosynthesis.
Intragenic suppression of gain‐of‐function mutations in the Escherichia coli mechanosensitive channel, MscL
Authors: Li Yuezhou , Wray Robin , Blount Paul 
Published: July 2004

SummaryMechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological ‘pressure release valves’. One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli. The E. coli MscL is a 136‐amino‐acid protein organized as a homopentamer with each subunit containing two transmembrane segments. Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain‐of‐function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions. Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations. The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp. Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop. In contrast, only one mutation is found in the amino‐terminal S1 domain, and none is found within the carboxyl‐terminal domain. Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.
Trypanosomatid histones
Authors: Alsford Sam , Horn David 
Published: July 2004

SummaryThe histones are responsible for packaging and regulating access to eukaryotic genomes. Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals. Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post‐translational modification and function within chromatin. Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N‐terminal ‘tail’ domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage. For example, histone H3 contains a highly conserved methylation substrate. Trypanosomatids also possess at least three variant histones. Among these is an orthologue of H2A.Z, a histone involved in protecting ‘active’ chromatin from silencing in yeast. Histones provide docking platforms for a variety of regulatory factors. The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation.
DNA‐binding specificity of AdpA, a transcriptional activator in the A‐factor regulatory cascade in Streptomyces griseus
Authors: Yamazaki Haruka , Tomono Ayami , Ohnishi Yasuo , Horinouchi Sueharu 
Published: July 2004

SummaryAdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A‐factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ‐1‐like dimerization domain at its N‐terminal portion and a DNA‐binding domain with two helix–turn–helix motifs at its C‐terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA‐binding sequence, 5′‐TGGCSNGWWY‐3′ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13–14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (−200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from −40 to −50 and from −50 to −60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13–14 bp and 2 bp. The DNA‐binding domain is therefore connected to the ThiJ/PfpI/DJ‐1‐like dimerization domain with a flexible linker. The DNA‐binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.
Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant
Authors: Yildiz Fitnat H. , Liu Xiaole S. , Heydorn Arne , Schoolnik Gary K. 
Published: July 2004

SummaryReversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the  rugose  and  smooth  variants  of  the  same  strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPSEl Tor, a rugose‐associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation‐specific gene expression. Bioinformatics analysis of these expression data shows that ‘rugosity’ and ‘smoothness’ are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.