- Volume 152, Issue 10, 2006
Volume 152, Issue 10, 2006
- Mini-Review
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Different subcellular locations of secretome components of Gram-positive bacteria
More LessGram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various locations in the cell. The translocons of the general Sec secretion system in the rod-shaped bacterium Bacillus subtilis have been shown to localize in spirals along the cytoplasmic membrane, whereas the translocons in the coccoid Streptococcus pyogenes are located in a microdomain near the septum. In both bacteria the Sec translocons appear to be located near the sites of cell wall synthesis. The Tat secretion system, which is used for the transport of folded proteins, probably localizes in the cytoplasmic membrane and at the cell poles of B. subtilis. In Lactococcus lactis the ABC transporter dedicated to the transport of a small antimicrobial peptide is distributed throughout the membrane. Possible mechanisms for maintaining the localization of these secretion machineries involve their interaction with proteins of the cytoskeleton or components of the cell wall synthesis machinery, or the presence of lipid subdomains surrounding the transport systems.
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- Biochemistry And Molecular Biology
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RepAM of the Amycolatopsis methanolica integrative element pMEA300 belongs to a novel class of replication initiator proteins
More LessAccessory genetic elements, such as plasmids and integrative elements, are widespread amongst actinomycetes, but little is known about their functions and mode of replication. The conjugative element pMEA300 from Amycolatopsis methanolica is present mostly in an integrated state at a single specific site in the chromosome, but it can also replicate autonomously. Complete nucleotide sequencing, in combination with deletion studies, has revealed that orfB of pMEA300 is essential for autonomous replication in its host. In this study, it was shown that purified OrfB protein binds specifically to the 3′ end of its own coding sequence. Within this short sequence, a putative hairpin structure is located, which contains several direct and inverted repeats, and a nucleotide stretch that resembles the nicking site of the pC194 family of rolling circle replicating plasmids. Additional binding studies revealed that OrfB binds to an 8 bp inverted repeat that occurs three times within the hairpin structure. The data presented show that OrfB is the replication initiator (Rep) protein of pMEA300, and is therefore termed RepAM. Surprisingly, RepAM lacks significant sequence similarity with known prokaryotic Rep proteins, but it is highly similar to a number of yet uncharacterized ORFs that are located on integrative and conjugative elements of other actinomycetes. It is concluded that RepAM and its homologues are members of a novel class of Rep proteins.
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The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin
Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Δ : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-β-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-β-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-β-glucan synthase.
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Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD
More LessThe molecular target for the bacteriolytic E protein from bacteriophage ϕX174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide Epep, containing the N-terminal transmembrane α-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of Epep was found by circular dichroism (CD) spectroscopy to be 100 % α-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, Epep was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC50 0.15 μM), implying a strong interaction between E and SlyD. Epep was found to inhibit E. coli MraY activity when assayed in membranes (IC50 0.8 μM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein–protein interaction is formed between E and MraY at a site within the transmembrane region.
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The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus
More LessThe biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr8, is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.
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Stimulation of the λ p R promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation
More LessEscherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage λ promoters p I, p aQ and p R. While SeqA-mediated stimulation of p I and p aQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p R remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p R transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p R was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p R transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.
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Complementation of daptomycin dptA and dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics
More LessDaptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as Cubicin® (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the ΔdptD mutation.
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- Environmental Microbiology
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A mannose-sensitive haemagglutinin (MSHA)-like pilus promotes attachment of Pseudoalteromonas tunicata cells to the surface of the green alga Ulva australis
This study demonstrates that attachment of the marine bacterium Pseudoalteromonas tunicata to the cellulose-containing surface of the green alga Ulva australis is mediated by a mannose-sensitive haemagglutinin (MSHA-like) pilus. We have identified an MSHA pilus biogenesis gene locus in P. tunicata, termed mshI1I2JKLMNEGFBACDOPQ, which shows significant homology, with respect to its genetic characteristics and organization, to the MSHA pilus biogenesis gene locus of Vibrio cholerae. Electron microscopy studies revealed that P. tunicata wild-type cells express flexible pili peritrichously arranged on the cell surface. A P. tunicata mutant (SM5) with a transposon insertion in the mshJ region displayed a non-piliated phenotype. Using SM5, it has been demonstrated that the MSHA pilus promotes attachment of P. tunicata wild-type cells in polystyrene microtitre plates, as well as to microcrystalline cellulose and to the living surface of U. australis. P. tunicata also demonstrated increased pilus production in response to cellulose and its monomer constituent cellobiose. The MSHA pilus thus functions as a determinant of attachment in P. tunicata, and it is proposed that an understanding of surface sensing mechanisms displayed by P. tunicata will provide insight into specific ecological interactions that occur between this bacterium and higher marine organisms.
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Ecophysiology of different filamentous Alphaproteobacteria in industrial wastewater treatment plants
The ecophysiology of five filamentous species affiliated to the Alphaproteobacteria was investigated in industrial activated sludge systems. The five species, ‘Candidatus Alysiosphaera europaea’, ‘Candidatus Monilibacter batavus’, ‘Candidatus Alysiomicrobium bavaricum’, ‘Candidatus Sphaeronema italicum’ and Meganema perideroedes, are very abundant in industrial wastewater treatment plants and are often involved in bulking incidents. The morphology of these filamentous bacterial species resembled Eikelboom's Nostocoida limicola, or Type 021N, and could only be correctly identified by using fluorescence in situ hybridization (FISH), applying species-specific gene probes. Two physiological groupings of the five species were found using microautoradiography combined with FISH. Group 1 (‘Ca. Monilibacter batavus' and ‘Ca. Sphaeronema italicum’) utilized many short-chained fatty acids (acetate, pyruvate and propionate), whereas Group 2 (‘Ca. Alysiosphaera europaea’, ‘Ca. Alysiomicrobium bavaricum’ and Meganema perideroedes) could also exploit several sugars, amino acids and ethanol. All species had polyhydroxyalkanoate granules present and several of the species had a very large storage capacity. No activity was found under strict anaerobic conditions, while uptake of substrate was observed in the presence of nitrate or nitrite as potential electron acceptor. However, for all species a reduced number of substrates could be consumed under these conditions compared to aerobic conditions. Only a little exo-enzymic activity was found and nearly all species had a hydrophobic cell surface. Based on knowledge of the ecophysiological potential, control strategies are suggested.
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Diversity of culturable halophilic sulfur-oxidizing bacteria in hypersaline habitats
More LessUnexpectedly high culturable diversity of moderately and extremely halophilic obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) was discovered in the sediments of various hypersaline habitats, including chloride-sulfate lakes in Mongolia, Russia and Ukraine, a sea saltern in Slovenia and a deep-sea salt brine from the Mediterranean. Six different groups of halophilic SOB, including four new genera, all belonging to the Gammaproteobacteria, were found. Two groups of moderately halophilic strictly aerobic SOB dominated at 2 M NaCl, including representatives of the genus Halothiobacillus (in fully aerobic conditions) and Thiomicrospira (in micro-oxic conditions). Under denitrifying conditions at 2 M NaCl, a group of moderately halophilic and facultatively anaerobic SOB was selected, capable of complete denitrification of nitrate. The group represents a new genus with closest relatives among as yet undescribed marine thiodenitrifying isolates. With thiocyanate as a substrate, an enrichment culture at 2 M NaCl yielded a pure culture of moderately halophilic SOB capable of aerobic growth with thiocyanate and thiosulfate at up to 4 M NaCl. Furthermore, this bacterium also grew anaerobically using nitrite as electron acceptor. It formed a new lineage distantly related to the genus Thiomicrospira. Enrichments at 4 M NaCl resulted in the domination of two different, previously unknown, groups of extremely halophilic SOB. Under oxic conditions, they were represented by strictly aerobic spiral-shaped bacteria, related to the Ectothiorhodospiraceae, while under denitrifying conditions a group of facultatively anaerobic nitrate-reducing bacteria with long rod-shaped cells was selected, distantly related to the genus Acidithiobacillus.
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Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103
More LessThe genes for polyethylene glycol (PEG) catabolism (pegB, C, D, A and E) in Sphingopyxis macrogoltabida strain 103 were shown to form a PEG-inducible operon. The pegR gene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putative σ 70-type promoter sites were identified in the pegB, pegA and pegR promoters. A promoter activity assay showed that the pegB promoter was induced by PEG and oligomeric ethylene glycols, whereas the pegA and pegR promoters were induced by PEG. Deletion analysis of the pegB promoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of the pegBCDAE operon. Gel retardation assays demonstrated the specific binding of PegR to the pegB promoter. Transcriptional fusion studies of pegR with pegA and pegB promoters suggested that PegR regulates the expression of the pegBCDAE operon positively through its binding to the pegB promoter, but PegR does not bind to the pegA promoter. Two specific binding proteins for the pegA promoter were purified and identified as a GalR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GalR/LacI-type regulator was found in the pegA and pegR promoters. These results suggested the dual regulation of the pegBCDAE operon through the pegB promoter by an AraC-type regulator, PegR (PEG-independent), and through the pegA and pegR promoters by a GalR/LacI-type regulator together with HU (PEG-dependent).
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- Genes And Genomes
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Functional analysis of 11 putative essential genes in Bacillus subtilis
More LessSystematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these (yacA, ydiB, ydiC, ykqC, ylaN, yloQ, ymdA, yneS, yqeI, yqjK and ywlC) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes (ydiB, yloQ, yqeI and ywlC) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.
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Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis
More LessThe soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a σ 54 promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the α subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site ( Prior & Dalton, 1985 ). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO− mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO− mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.
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Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis
More LessMembers of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and its cognate response regulator (RR). With the use of in silico techniques, a complete set of HKs and RRs was recovered from eight completely sequenced B. cereus group genomes. By applying a bidirectional best-hits method combined with gene neighbourhood analysis, a footprint of these proteins was made. Around 40 HK-RR gene pairs were detected in each member of the B. cereus group. In addition, each member contained many HK and RR genes not encoded in pairs (‘orphans’). Classification of HKs and RRs based on their enzymic domains together with the analysis of two neighbour-joining trees of these domains revealed putative interaction partners for most of the ‘orphans’. Putative biological functions, including involvement in virulence and host–microbe interactions, were predicted for the B. cereus group HKs and RRs by comparing them with those of B. subtilis and other micro-organisms. Remarkably, B. anthracis appeared to lack specific HKs and RRs and was found to contain many truncated, putatively non-functional, HK and RR genes. It is hypothesized that specialization of B. anthracis as a pathogen could have reduced the range of environmental stimuli to which it is exposed. This may have rendered some of its TCSs obsolete, ultimately resulting in the deletion of some HK and RR genes.
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The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions
Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor–regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.
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Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger
As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose- or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose- (sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a ΔcreA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the ΔcreA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger.
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Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390
The production of Staphylococcus aureus virulence factors is under the control of complex regulatory circuits. Most studies aimed at defining these regulatory networks have focused on derivatives of the strain NCTC 8325, most notably RN6390. However, all NCTC 8325 derivatives, including RN6390, possess an 11 bp deletion in rsbU. This deletion renders NCTC 8325 derivatives naturally sigma-factor-B deficient. Recent studies have shown that RN6390 is also deficient, in comparison to clinical isolates, with respect to biofilm formation, a process which is important for both pathogenesis and antimicrobial resistance. Based on these considerations, the authors carried out genome-scale transcriptional profiling, comparing RN6390 with the virulent rsbU-positive clinical isolate UAMS-1. The results revealed significant genome-wide differences in expression patterns between RN6390 and UAMS-1, and suggested that the overall transcriptional profile of UAMS-1 is geared toward expression of factors that promote colonization and biofilm formation. In contrast, the transcriptional profile of RN6390 was heavily influenced by RNAIII expression, resulting in a phenotype characterized by increased production of exoproteins, and decreased capacity to form a biofilm. The greater influence of agr in RN6390 relative to UAMS-1 was also evident when the transcriptional profile of UAMS-1 was compared with that of its isogenic sarA and agr mutants. Specifically, the results indicate that, in contrast to NCTC 8325 derivatives, agr plays a limited role in overall regulation of gene expression in UAMS-1, when compared with sarA. Furthermore, by defining the sarA regulon in a biofilm-positive clinical isolate, and comparing the results with transcriptional profiling experiments defining biofilm-associated gene expression patterns in the same strain, the authors identified a sarA-regulated operon (alsSD) that is also induced in biofilms, and demonstrated that mutation of alsSD results in reduced capacity to form a biofilm.
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- Pathogens And Pathogenicity
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Amino acids mediate colony and cell differentiation in the fungal pathogen Candida parapsilosis
More LessCandida parapsilosis is responsible for severe cases of non-albicans systemic candidiasis and is one of the leading causes of mortality in neonates. The molecular mechanisms underlying this organism's virulence remain unknown. Unlike C. albicans, which can exist in several morphogenetic forms, C. parapsilosis exists in either the yeast or pseudohyphal forms. The environmental signals that trigger pseudohyphal differentiation and the signalling pathways that transduce these signals are unknown. This paper provides evidence for the role of amino acids in morphogenesis in C. parapsilosis. The cell and colony morphologies, pseudohyphal differentiation and invasive growth of five C. parapsilosis isolates were characterized in ammonium-rich minimal media lacking or supplemented with naturally occurring amino acids. C. parapsilosis underwent dramatic changes in cellular and colony morphology and formed pseudohyphae in response to a specific subset of amino acids. Transport studies showed that these amino acid inducers activate the transport of some, but not all, unrelated amino acids. Interestingly, citrulline, an amino acid that is not transported in the presence of ammonium, strongly induced pseudohyphal morphogenesis in C. parapsilosis under these conditions. Together the data suggest that amino acids are important morphogens in C. parapsilosis and that amino-acid-mediated morphogenesis in this organism does not require transport of the ligand across the plasma membrane.
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Inhibitors of Helicobacter pylori ATPase Cagα block CagA transport and cag virulence
With the steadily increasing occurrence of antibiotic resistance in bacteria, there is a great need for new antibacterial compounds. The approach described here involves targeting virulence-related bacterial type IV secretion systems (TFSSs) with small-molecule inhibitors. The cag TFSS of Helicobacter pylori was chosen as a model, and novel inhibitors directed against the cag VirB11-type ATPase Cagα were identified. The cag genes encode proteins that are components of a contact-dependent secretion system used by the bacterium to translocate the effector molecule CagA into host cells. Translocated CagA is associated with severe gastritis, and carcinoma. Furthermore, functional TFSSs and immunodominant CagA play a role in interleukin (IL)-8 induction, which is an important factor for chronic inflammation. Inhibitors of Cagα were identified by high-throughput screening of chemical libraries that comprised 524 400 small molecules. The ATPase activity of Cagα was inhibited by the selected compounds in an in vitro enzymic assay using the purified enzyme. The most active compound, CHIR-1, reduced TFSS function to an extent that cellular effects on AGS cells mediated by CagA were virtually undetectable, while reduced levels of IL-8 induction were observed. Gastric colonization by CHIR-1-pre-treated bacteria was found to be impaired in a dose-dependent manner using a mouse model of infection. Small-molecule Cagα inhibitors, the first described inhibitors of a TFSS, are potential candidates for the development of new antibacterial compounds that may lead to alternative medical treatments. The compounds are expected to impose weak selective pressure, since they target virulence functions. Moreover, the targeted virulence protein is conserved in a variety of bacterial pathogens. Additionally, TFSS inhibitors are potent tools to study the biology of TFSSs.
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A truncated Bacillus subtilis dal gene with a 3′ ssrA gene tag regulates the growth and virulence of racemase-deficient Listeria monocytogenes
More LessListeria monocytogenes (Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dal and dat) required for d-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenous d-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologous Bacillus subtilis dal gene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with an ssrA tag at the 3′-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew without d-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence of d-alanine. Addition of an ssrA tag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools of d-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-type L. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenous d-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.
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Volumes and issues
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Volume 103 (1977)
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Volume 102 (1977)
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Volume 101 (1977)
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Volume 100 (1977)
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Volume 99 (1977)
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Volume 98 (1977)
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Volume 97 (1976)
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Volume 96 (1976)
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Volume 95 (1976)
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Volume 94 (1976)
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Volume 93 (1976)
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Volume 92 (1976)
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Volume 91 (1975)
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Volume 90 (1975)
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Volume 89 (1975)
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Volume 88 (1975)
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Volume 87 (1975)
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)