- Volume 150, Issue 3, 2004
Volume 150, Issue 3, 2004
- Microbiology Comment
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- Cell And Developmental Biology
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Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells
More LessBdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.
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- Biochemistry And Molecular Biology
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The fifth gene of the iol operon of Bacillus subtilis, iolE, encodes 2-keto-myo-inositol dehydratase
The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as d-2,3-diketo-4-deoxy-epi-inositol.
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Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis
More LessBacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins – CheC, CheD and CheV – mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type. This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type. Removal of all known adaptation systems – the methylation, CheC and CheV systems – resulted in a mutant (cheRBCDV) that still retained some adaptation following the addition of attractant.
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Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors
More LessExtracytoplasmic function (ECF) sigma factors constitute a diverse family of proteins, within the class of the sigma 70 subunit of RNA polymerase. Most members of the family studied to date are known to regulate gene expression in response to stress conditions. The Bacillus subtilis genome encodes at least 17 distinct sigma factors, seven of which are members of the ECF subfamily. Among these, five sigma factors, namely SigV, SigW, SigX, SigY and SigM, are encoded by the first genes of the cognate sigma operons. Disruption or repressed expression of the downstream gene(s) resulted in transcriptional activation of the cognate sigma operon. Moreover, in vivo protein–protein interaction analyses by yeast two-hybrid experiments indicated that these immediate downstream gene products bind the cognate ECF sigma factor, suggesting that they function as anti-sigma factors by capturing sigma factor on the inner surface of the cytoplasmic membrane. Interaction with other sigma factors was not observed. The results presented here also show that these anti-sigma factors interact with ECF sigma factors through their N-terminal region, implying that the N-terminal domain resides inside the cytoplasmic membrane.
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Complementation of a ΔccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains
In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.
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Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri
More LessLactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with β(2→6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with β(2→1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (LevΔ773His) that produced the same levan polymer [with only 2–3 % β(2→1→6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro. Lev is strongly dependent on Ca2+ ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 °C, kinetics that are best described by the Hill equation.
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RppA, a transducer homologue, and MmrA, a multidrug transporter homologue, are involved in the biogenesis and/or assembly of polysaccharide in Myxococcus xanthus
More LessMyxococcus xanthus cells move by gliding, and form multicellular fruiting bodies under conditions of starvation. The authors cloned a gene, designated rppA (for receptor for polysaccharide production), which encodes a methyl-accepting protein homologous to the chemotaxis transducers in eubacteria. The rppA gene was co-transcribed with mmrA, a gene homologous to various multidrug transporter genes. The rppA or mmrA single mutants showed almost identical phenotypes to the wild-type strain; however, the rppA-mmrA double mutant exhibited reduced colony expansion, cell–cell agglutination and cellular reversal frequency. The double-mutant cells also showed less binding to Congo red, which mainly binds to fibril polysaccharide, than wild-type cells. Analysis of total polysaccharide in stationary-phase cells demonstrated that in the double mutant, polysaccharide levels were decreased by about 30 % as compared with the wild-type strain. These results indicated that RppA and MmrA play a role in the biogenesis and/or assembly of polysaccharide, and the phenotypes of the double mutant may be due to the reduction in fibril polysaccharide.
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Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489
More LessThe biosynthetic gene clusters of the gyrase inhibitors coumermycin A1 and clorobiocin contain two different resistance genes (gyrB R and parY R). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and GyrBR was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and ParYR catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.
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Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021
More LessSequencing of the Sinorhizobium meliloti strain 1021 genome led to the detection of 6204 open reading frames, 41 % of which have no hypothetical function. To help annotate this genome, a transcriptome analysis was carried out with a dedicated microarray consisting of 146 genes belonging to three different classes: (i) no hypothetical function; (ii) potentially involved in oxidative stress responses; (iii) known to participate in oxidative stress responses (e.g. catalase and superoxide dismutase genes). This transcriptome analysis, together with biological experiments and in silico investigations, identified new genes induced by exogenous H2O2. The smc01944 gene was the most strongly induced: quantitative PCR showed that the amount of smc01944 mRNA increased 50-fold following the addition of 10 mM H2O2, whereas the amount of katA mRNA (encoding a catalase) only increased 10-fold. Smc01944 is a non-haem chloroperoxidase (Cpo). The only member of this family to have been so far characterized is encoded by prxC of Pseudomonas fluorescens. Unexpectedly, the NH2-terminus of Smc01944 includes a signal peptide and Smc01944 is secreted into the supernatant. Interestingly, smc01944 is preceded by smc01945, encoding an OhrR-like regulator (MarR family). Thus, Smc01944 is the first exported Cpo encoded by a gene possibly regulated by an OhrR regulator. It was also shown that smc01944 is induced by t-butyl and cumene hydroperoxides but only slightly by menadione. The study of Smc01944 described in this work showed that the oxidative stress response of S. meliloti seems to differ from that of other bacteria characterized to date.
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Expression of the Pneumocystis carinii major surface glycoprotein epitope is correlated with linkage of the cognate gene to the upstream conserved sequence locus
More LessThe major surface glycoprotein (MSG) is a variable surface antigen of the pathogenic fungus Pneumocystis carinii. Many forms of MSG are encoded by a gene family. Expression of the MSG gene family is believed to be controlled in a cis-dependent fashion. Transcription of a given MSG gene is correlated with linkage of that gene to a unique locus called the upstream conserved sequence (UCS). These data predict that the MSG protein on a given organism will match that encoded by the MSG gene at the UCS locus in that organism. To test this hypothesis, a monoclonal antibody (mAb) that recognizes a small number of MSG isoforms was identified, and the DNA sequence encoding the mAb epitope (epitope-encoding sequence, EES) was determined. Western blotting, immunofluorescence and DNA hybridization showed that expression of the mAb epitope was associated with the presence of the EES at the UCS locus. Correlation of epitope expression and UCS linkage supports the hypothesis that expression of a particular MSG on the surface requires UCS linkage of the gene encoding it.
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Temperature-dependent processing of the cspA mRNA in Rhodobacter capsulatus
More LessThe expression of genes for cold-shock proteins is proposed to be regulated primarily at the post-transcriptional level by increase of mRNA stability after transition to low temperatures. Destabilization of the Escherichia coli cold-induced cspA transcript at 37 °C as well as stabilization upon cold shock is known to depend on the unusually long (159 nt) 5′-untranslated region. Determination of the cspA mRNA 5′-end from Rhodobacter capsulatus revealed a shorter distance between the start of transcription and the start codon for translation. The cspA mRNA of R. capsulatus was shown to be stabilized at low temperatures to a greater extent than other investigated transcripts. To address the mechanism of decay of the cspA transcript, it was incubated with purified degradosome of R. capsulatus. Endoribonucleolytic in vitro cleavage in the 5′-untranslated region as reported for the cspA transcript of E. coli in vivo was not observed. Instead, the data indicated that the cspA mRNA decay in R. capsulatus is mediated by endoribonucleolytic cleavages within the cspA coding region.
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Redox property and regulation of PpsR, a transcriptional repressor of photosystem gene expression in Rhodobacter sphaeroides
More LessPpsR from Rhodobacter sphaeroides is involved in the repression of photosystem gene expression. The PpsR protein was heterologously overexpressed and purified to homogeneity. Gel mobility shift assay showed that the purified PpsR has DNA-binding activity. SDS-PAGE analysis showed that some portions of PpsR were oxidized, indicating that intramolecular or intermolecular disulphide bonds were formed between the two cysteines in each subunit. When the disulphide bond of PpsR was reduced by DTT, the binding activity of PpsR to the puc promoter region distinctly increased. The changes in protein level and DNA-binding activity of PpsR were observed in a conjugant with an extra copy of the ppsR gene and in a PpsR-null mutant (PPS1), respectively. Both cysteines in PpsR existed in their reduced form under aerobic, anaerobic-dark and anaerobic-light growth conditions, as determined using thiol-specific chemical modification. In an AppA-null mutant (APP11), the binding activity and the amount of PpsR decreased compared to those of the wild-type and an appA-complemented strain, and decreased even more under anaerobic-dark conditions than under aerobic conditions. PpsR had a redox-sensitive property but retained its reduced state in the cell, and its amount was reduced by disruption of AppA.
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Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase
In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)+-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an α 2 β 2 arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the α 2 β 2 tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)+-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)+-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation.
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- Biodiversity And Evolution
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Molecular phylogeny, biogeography and speciation of the mushroom species Pleurotus cystidiosus and allied taxa
More LessMembers of the mushroom genus Pleurotus form a heterogeneous group of edible species of high commercial importance. Subgenus Coremiopleurotus includes taxa that produce synnematoid fructifications (anamorphic state). Several species, subspecies and varieties have been described in Coremiopleurotus. These taxa are discriminated by minute morphological differences and correspond to Pleurotus cystidiosus sensu lato. A worldwide geographical sampling of Coremiopleurotus taxa and nucleotide sequence data from the internal transcribed spacer of the nuclear rRNA genes (ITS) were used to produce a molecular phylogeny for the group. Also conducted were new interfertility studies, and a summary of the mating data currently available in the literature is provided. Both ITS phylogeny and mating data supported the distinction between Pleurotus australis (a species apparently endemic to New Zealand and Australia) and P. cystidiosus sensu lato. Within P. cystidiosus sensu lato, ITS phylogeny showed a deep split between Old and New World isolates and clearly distinguished four distinct clades that strongly corresponded to the geographical origin of the strains. In the Old World, one clade is composed of isolates from Europe and Africa, and one clade is composed of isolates from Asia (including collections from Hawaii). In the New World, one clade is restricted to isolates from Mexico, and one clade includes all the authors' North America isolates, one collection from Japan and one collection from South Africa. Mating data revealed a high level of interfertility among strains of P. cystidiosus sensu lato, except that isolates from Mexico were nearly fully intersterile with the other collections. Nucleotide sequence divergence in the ITS1–5·8S rDNA–ITS2 regions among intercompatible P. cystidiosus collections was very high (0–6·9 %) in comparison to that reported in other biological species of basidiomycetes (0–3 %), indicating significant genetic divergence between geographically isolated populations of the P. cystidiosus group. The phylogenetic species concept, as well as molecular, mating and geographical evidence, was used to recognize five species in the subgenus Coremiopleurotus: P. australis (in New Zealand and Australia), Pleurotus abalonus (in Asia and Hawaii), Pleurotus fuscosquamulosus (in Africa and Europe), Pleurotus smithii (in Mexico) and Pleurotus cystidiosus sensu stricto (in North America). However, geographical boundaries between these species are not strict, as rare events of long distance dispersal have occurred.
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- Environmental Microbiology
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Groupings of highly similar major surface protein (p44)-encoding paralogues: a potential index of genetic diversity amongst isolates of Anaplasma phagocytophilum
Anaplasma phagocytophilum is a tick-borne bacterium that is zoonotic in the USA and southern Europe, but although the bacterium is endemic in the UK, no cases of clinical human disease have yet been detected in that country. Potential genomic differences amongst UK and USA isolates were investigated by comparing partial 16S rRNA gene and p44 paralogue sequences amplified by PCR from 10 UK ruminant or tick isolates, with published sequences from USA isolates. No significant clustering among the isolates was resolved by phylogenetic analysis of alignments containing 16S rRNA gene sequences. The structure of predicted proteins encoded by p44 paralogues, amplified from 81 clones obtained from the UK isolates, was similar to that described previously for paralogues from USA isolates. Paralogue sequences did not obviously cluster by country, host species or isolate, but most paralogues were 30–70 % similar, making meaningful alignments difficult. Some p44 paralogues from different isolates formed clusters of sequences that were more than 90 % similar to one another (‘similarity groups’). The paralogues in each cluster were particularly similar in gene regions most likely to code for ligands. In the sample studied, 95 % of the similarity groups comprised paralogues from either USA or UK isolates only and occurred with greater frequency amongst paralogues from USA rather than UK isolates. These findings raise the hypothesis that sequences of paralogues in similarity groups may provide an index of adaptation of different ‘strains' of A. phagocytophilum to specific reservoir hosts in different geographical locations, and any associations with infectivity for different species including humans.
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- Genes And Genomes
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Two distinct types of rRNA operons in the Bacillus cereus group
More LessThe Bacillus cereus group includes insecticidal bacteria (B. thuringiensis), food-borne pathogens (B. cereus and B. weihenstephanensis) and B. anthracis, the causative agent of anthrax. The precise number of rRNA operons in 12 strains of the B. cereus group was determined. Most of the tested strains possess 13 operons and the tested psychrotolerant strains contain 14 operons, the highest number ever found in bacteria. The separate clustering of the tested psychrotolerant strains was confirmed by partial sequencing of several genes distributed over the chromosomes. Analysis of regions downstream of the 23S rRNA genes in the type strain B. cereus ATCC 14579 indicates that the rRNA operons can be divided into two classes, I and II, consisting respectively of eight and five operons. Class II operons exhibit multiple tRNA genes downstream of the 5S rRNA gene and a putative promoter sequence in the 23S–5S intergenic region, suggesting that 5S rRNA and the downstream tRNA genes can be transcribed independently of the 16S and 23S genes. Similar observations were made in the recently sequenced genome of B. anthracis strain Ames. The existence of these distinct types of rRNA operons suggests an unknown mechanism for regulation of rRNA and tRNA synthesis potentially related to the pool of amino acids available for protein synthesis.
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Characterization of a new internal promoter (P3) for Rhizobium leguminosarum hydrogenase accessory genes hupGHIJ
More LessSynthesis of the Rhizobium leguminosarum [NiFe] hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL). Transcription of hupSL is controlled by a −24/−12-type promoter (P1), located upstream of hupS and regulated by NifA. In this work, a second −24/−12-type promoter (P3), located upstream of the hupG gene and transcribing hupGHIJ genes in R. leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster. Promoter P3 was also active in R. leguminosarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants. Both NifA and NtrC activated P3 expression in the heterologous host Klebsiella pneumoniae. Also, P3 activity was highly stimulated by K. pneumoniae NifA in Escherichia coli. This NifA activation of P3 expression only required the σ 54-binding site, and it was independent of any cis-acting element upstream of the σ 54 box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme. P3-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern. However, P3 was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at P1. This fact and the lack of an activator recruitment system suggest that P3 plays a secondary role in symbiotic hupGHIJ expression.
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- Pathogens And Pathogenicity
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Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin
Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic ‘attaching and effacing’ (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin. BFP are involved in bacteria–bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA + espA + eae +) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA + espA − eae −) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA − espA + eae −) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA − espA − eae +), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA + espA + eae −) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA + espA + eae −) and UMD872 (bfpA + espA − eae +) showed adherence characteristics of strain UMD880 (bfpA + espA − eae −), whilst triple mutant strain UMD888 (bfpA − espA − eae −) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.
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The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes
More LessInvasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of P. aeruginosa was screened to identify P. aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the fimV gene. A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility. A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.
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Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species
More LessThe aetiological agent of Lyme disease, Borrelia burgdorferi cycles between its tick vector and mammalian hosts, implying that it can sense different environments and consequently change the expression of genes encoding several surface-associated proteins. The genome of the type strain B. burgdorferi B31 has revealed 175 different gene families. The p13 gene, situated on the chromosome, encodes a channel-forming protein that belongs to the gene family 48 consisting of eight additional paralogous genes. The heterogeneity of the P13 protein from different Lyme disease Borrelia strains was investigated. The predicted surface-exposed domains are the most heterogeneous regions and contain probable epitopes of P13. The membrane-spanning architecture of P13 was determined and a model for the location of this protein in the outer membrane is presented. The transcription of the paralogues of gene family 48 during in vitro culturing and in a mouse infection model was also analysed. The bba01 gene is the only p13 paralogue present in all three Lyme-disease-causing genospecies; it is stable during cultivation in vitro and the BBA01 protein was expressed in all Borrelia strains investigated. Conversely, paralogues bbi31, bbq06 and bbh41 were only detected in B. burgdorferi and the corresponding plasmids harbouring bbi31 and bbh41 were lost during in vitro passage. Finally, p13 and bbi31 are the only members of gene family 48 that are transcribed in mice, suggesting their importance during mammalian infection.
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Campylobacter jejuni infection of differentiated THP-1 macrophages results in interleukin 1β release and caspase-1-independent apoptosis
More LessApoptosis induction of host macrophages has emerged as a common virulence mechanism among bacterial pathogens. Infection with Campylobacter jejuni is a leading cause of gastroenteritis worldwide and is characterized by an acute inflammatory response in the small intestine. The authors used the human monocytic cell line THP-1 to examine apoptosis induction and pro-inflammatory cytokine production during C. jejuni infection. Flow cytometric analysis revealed that 48 h after inoculation, a C. jejuni wild-type isolate induced apoptosis in 63 % of THP-1 cells while only 34 % of cells inoculated with a ciaB mutant, which does not secrete the Cia (Campylobacter invasion antigens) proteins, underwent apoptosis. Complementation of the ciaB mutant resulted in levels of apoptosis similar to those induced by the C. jejuni wild-type isolate, suggesting that the Cia proteins have a role in apoptosis induction. It was shown that a proteinase K- and heat-stable component of C. jejuni also stimulated THP-1 apoptosis. Inoculation with a C. jejuni gmhD mutant indicated that lipooligosaccharide was not the stimulatory molecule. Immunoblot and ELISA analyses revealed that C. jejuni infection stimulated the synthesis, processing and secretion of interleukin 1β (IL-1β). Inhibition of caspase 1 activity eliminated IL-1β processing and secretion, but did not affect apoptosis induction. In addition, treatment of cells with a caspase-9-specific inhibitor did not affect apoptosis induction, arguing against activation of an apoptotic pathway dependent on either caspase 1 or 9 activation. Collectively, these data suggest that the inoculation of macrophages with C. jejuni results in the processing of IL-1β and apoptosis through different regulatory pathways. Furthermore, these data argue that C. jejuni may use a mechanism distinct from Salmonella typhimurium and Shigella flexneri to initiate macrophage apoptosis and release of IL-1β.
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- Physiology
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Effect of acid stress on the physiology of biofilm cells of Streptococcus mutans
More LessStreptococcus mutans is a component of the dental plaque biofilm and an important aetiological agent in dental caries. Although this organism growing in the suspended (planktonic) state has been well characterized, relatively little is known about its physiology in biofilms, particularly in the acidic environments associated with caries development. The authors determined the effect of biofilm age (1–5 days) and cell density on selected metabolic properties under conditions of glucose limitation in a biofilm-chemostat at pH 7·5 and compared these baseline values with those of 3 day biofilms subjected to acid stress. Biofilm cell biomass more than doubled over the 5 day experimental period under baseline conditions, with the glycolytic rate, glucose uptake, glucose-PTS (phosphotransferase system) activity and protein synthesis maximum at 1–2 days. DNA and RNA synthesis increased for the first 3 days before decreasing in the 5 day biofilms, while H+/ATPase activity was higher in 5 day biofilms than 1 day biofilms, with overall activity 5–13-fold higher per cell unit than in the associated planktonic cells. Glucose pulsing (50 mM final concentration) for three consecutive days without pH control for 5 h (pH 4·39±0·02) resulted in a progressive decrease in planktonic cell numbers; however, the rate of acid formation and glucose utilization in the chemostat by these cells increased per cell unit. Assays for carbohydrate metabolism in the latter cells showed increased activity, as did an assay for H+/ATPase (8-fold); however, DNA, RNA and protein synthesis were repressed (0·3–0·7-fold). Although the 3 day biofilm viable cell counts declined by 51 % on glucose pulsing, all the physiological parameters measured by cell unit increased in activity, with notable increases in RNA and protein synthesis (4·6–7·6-fold). The results indicate that the maintenance of intracellular pH homeostasis is the basis of the enhanced physiological status and acid tolerance of biofilm cells.
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Disruption of the gene encoding the V-ATPase subunit A results in inhibition of normal growth and abolished sporulation in Aspergillus nidulans
More LessThe authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn2+ concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.
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