- Volume 144, Issue 11, 1998
Volume 144, Issue 11, 1998
- Review Article
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- Antigens And Immunity
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Immunization with recombinant class I outermembrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci
More LessSUMMARY: The porA gene from Neisseria meningitidis was cloned into the pRSETA vector and recombinant class 1 outer-membrane protein expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography on a Ni2+ matrix and used for immunization of mice with conventional AI(OH), adjuvant, with experimental adjuvants which have the potential for human use, and with liposomes. The resulting sera were analysed for the magnitude, subclass distribution and antigenic specificity of the immune response. In addition, surface plasmon resonance (SPR) was used to quantify antibody avidity by analysis of the kinetics of binding to native class 1 protein. Immunization withconventional and experimental adjuvants induced antibodies of low avidity that did not recognize native class 1 protein. In contrast, immunization with recombinant protein inliposomes induced antibodies of high avidity which recognized native class 1 protein, as measured by their ability t o label meningococcal cells in immunofluorescence assays and to inhibit the binding of a protective mAb. These properties were associated with the presence in sera of high levels of antibodies with the ability t o induce complement-mediated killing of meningococci. These data show that liposomes containing recombinant class 1 protein represent a potential basis of future vaccines, of defined composition, designed for the prevention of group B meningococcal infections.
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- Development And Structure
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A Candida albicans chaperonin subunit (CaCct8p) as a suppressor of morphogenesis and Ras phenotypes in C. albicans and Saccharomyces cerevisiae
More LessSUMMARY: Saccharomyces cerewisiae and the pathogen Candida albicans can be induced to undergo morphogenesis from a yeast to a filamentous form. A C. albicansgene (CaCCT8) was identified encoding a subunit of the Cct chaperonin complex, whose expression prevents filament formation in both fungi without interfering with growth of the yeast form. In 5. cerewisiae, pseudohyphal growth induced by Ra2 119va, by overproduction of Phdlp or by expression of the C. albicans EFGl gene, was blocked by CaCct8p and its N-terminally deleted derivative CaCct8-Alp; in contrast, pseudohyphal induction by othe components (Cphlp, Cdc42p) could not be suppressed, indicating that morphogenesis per se is not inhibited. CaCCT8 expression also interfered with other Ra2p va119, phenotypes, including heat sensitivity, lack of glycogen accumulation and lack of sporulation. In C. albicans, overproduction of CaCct8p effectively blocked hyphal morphogenesis induced by starvation conditions and by serum. The results suggest that the activity of a component in the Ras2p signal transduction pathway is suppressed by excess chaperonin subunits. This component may be a novel folding target for the Cct complex. In agreement with this hypothesis, disruption of one of the two CaCC7'8 alleles in C. albicans led t o defective hyphal morphogenesis.
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ldentif ication of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom)
More LessSUMMARY: Three proteins with characteristic features of class I hydrophobins, designated POH1, POH2 and POH3, were isolated from the basidiomycete PIeurotus ostreatus. Based on N-terminal sequence analyses, their cDNAs were isolated using RT-PCR; the cDNAs and corresponding genes were sequenced and their regulation studied. POHI is expressed in the fruiting bodies but not in vegetative mycelium. The regulation of fWf2 and poH3 is tightly correlated. Both genes are switched off in the fruiting bodies but abundantly expressed in the vegetative mycelium of both monokaryon and dikaryon. POH2 and POH3 were isolated from the culture medium and from aerial hyphae. Co-purified POH2 and POH3 assembledin witro into a protein membrane with a typical rodlet pattern as found previously withother hydrophobins. Similar structures were detected on the surface of aerial hyphae.
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- Environmental Microbiology
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Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase
More LessSUMMARY: Biof ilm bacteria Enterobecter agglomerans 53b and Serratia marcescens Serr were isolated from a food processing factory. A bacteriophage (SF153b), which could infect and lyse strain 53b, was isolated from sewage. This has been shown to possess a polysaccharide depolymerase enzyme specific for the exopolysaccharide (EPS) of strain 53b. Using batch culture and chemostat-linked Modified Robbins Device systems it was observed that SF1 53b could degrade the EPS of a mono-species biofilm (strain 53b) and infect the cells. The disruption of the biofilm by phage was a combination of EPS degradation by the depolymerase and infection and subsequent cell lysis by the phage. Strain Serr biofilms were not susceptible to the phage and the biofilm EPS was not degraded by the phage glycanase, with the result that the biofilm was unaffected by the addition of SF153b phage. Scanning electron microscopy confirmed that specific phage could extensively degrade susceptible biofilms and continue to infect biofilm bacteria whilst EPS degradation was occurring.
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- Genetics And Molecular Biology
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Candida albicans SSDl can suppress multiple mutations in Saccharomyces cere visiae
More LessSUMMARY: The SSD1 gene of Saccharomyces encodes a 160 kDa cytoplasmic protein that can suppress mutations in a number of other genes. A functional homologue of SSD1 from the humanpathogen Candida albicans was isolated on the basis of its ability to restore viabilityat the restrictive temperature in a Saccharomyces cerevisiae swi4 ssdl-d strain. The C. albicans gene, designated CaSSD1, encodes a 1262 aa protein which has 47% identity overall to S. cerevisiae SSDI as well as significant identity to Schizosaccharomyces pombe dis3 and sts5 products. It is shown that CaSSD1 expression is constitutive through the mitotic cell cycle, which is consistent with a role for the protein in cell growth. CaSSD1 rescues the swiP defect in an ssd1-d background when expressed from its own promoteron a single-copy plasmid and under the same conditions can rescue mutations in genes encoding protein phosphatase type 2A catalytic subunits. These data suggest that CaSSD1, like its S. cerevisiae homologue, can limit the effect of mutations on a variety of cellular processes.
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Molecular and biochemical analysis of a 105 kDa Mycoplasma gallisepticum cytadhesin(GapA)
More LessSUMMARY: The identification of a gene (g8pA) from Mycoplasma gallisepticum with homology to the P I cytadherence gene of Mycoplasma pneumoniae is reported. The gapA gene is a 28 pORF encoding a protein with a molecular mass of 105 kDa. Nucleotide sequence analysis of the gapA gene revealed 45% homology t o the M. pneumoniae P I gene, 46% homology t o the Mycoplasma genitalium MgPa gene and 47% homology to the Mycoplasma pirum PI-like protein gene. It has a 64 mol% A+T content compared to 46,60 and 72 mol%respectively for the PI, MgPa and the Pl-like protein genes. As with the PI and MgPa genes, gapA is a central gene in a multi-gene operon, but unlike the P1 and MgPa genes, there is only a single copy of gapA in the genome. GapA is a trypsin-sensitive surface-exposed protein. Chicken tracheal-ring inhibition-of-attachment assays, using anti-GapA Fab fragments, resulted in 64% inhibition of attachment. These results indicated that GapA plays a rolein cytadherence of M. gallisepticum to host cells.
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Truncation as a novel form of variation of the p50 gene in Mycoplasma hominis
More LessSUMMARY: A characteristic feature of the mycoplasmas is the presence of variable surface proteins which may play an important role in the adaptation of the cell-wall-less organisms to their host environments. In addition, this antigen variation may be an important pathogenic property of the organism. The ubiquity of the gene encoding P50, an adhesin of Mycop/asrna hominis FBG, and its transcription were analysed in different isolates ofM. horninis. The p50 gene was present in all isolates tested. Based on Southern blot analysis and sequencing of the gene, the isolates could be classified into one of three distinct groups. Within two groups specific truncations of the p50 gene occurred. The reduction of the gene size was confirmed in Northern blot analysis of representative isolates from each group, with a decrease in transcript length from 1.6 kb in group G-1 down to 076 kb in group 6-3. In addition to truncation, a coincidental duplication of some gene segments was detected. This work has provided evidence for the genetic basis of a further variation in the M. horninis P50 adhesin.
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Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination
More LessSUMMARY: Electrotransformation has been used as a tool to introduce genes carried on replicative vectors in hundreds of bacterial species. In this study, the technique was used to try to obtain recombination of markers in the chromosome of the natura IIy transformable bacterium Streptococcus pneumoniae. Recombination was not observed even using naturally competent cultures. Both chromosomal and cloned DNA, denatured or native, were without effect. These results suggest that it is not sufficient to introduce DNA into the cell to obtain recombinants in this bacterium. The integration of markers into the chromosome in naturally competent cells must require DNA processing during entry. Electrotransformation of replicating plasmids is red-independent but can be facilitated by a red-dependent process. This facilitation required the induction of the natural competence machinery, probably involving partial homologous pairing.
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Streptococcus pyogenes protein F promotes invasion of HeLa cells
More LessSUMMARY: Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using 111916 insertion mutagenesis or an isogenic 5. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with 5. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did notpermit internalization of the E. coli cells.
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The kdgRKAT operon of Bacillus subtilis:detection of the transcript and regulation by the kdgR and ccpA genes
More LessSUMMARY: Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied. The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxyg I uconate kinase; kdgA, encoding 2-keto-3-deoxyg luconate-6-phosphate aldolase; and kdg, encoding a transporter. These four genes are organized in one transcriptional unit and map at 198" of the B. subtik chromosome. Primer extension experiments and Northern blot analysis show that an active σ-dependent promoter precedes kdgR and transcription is terminated at the putative pindependent terminator downstream of kdgr. The operon is negatively regulated by the kdgR and ccpA gene products, which belong t o the Lac1 family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.
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Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling
SUMMARY: The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase E and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-l inked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The a,s,eand -like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by -poly(~-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase E and a 135 kDa -like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei at growth arrest.
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A Mycobacterium tuberculosis operon encoding ESAT=6 and a novel low-molecular-mass culture filtrate protein (CFP-10)
More LessSUMMARY: The early secreted antigenic target 6 kDa protein (ESAT-6) is a potent T-cell protein antigen synthesized by Mycobacterium tuberculosis. Its corresponding gene (esat-6) is located in RD1, a 10 kb DNA region deleted in the attenuated tuberculosis vaccine strain Mycobacterium bowis BCG. The promoter region of M. tuberculosis esat-6 was cloned and characterized. A new gene, designated lhp and cotranscribed with esat-6, was identified. Moreover, computer searches in the M. tuberculosis genome identified 13 genes related to the lhplesat-6 operon, defining a novel gene family. The transcription initiation sites of the lhplesat-6 operon were mapped using M. tuberculosis RNA. The corresponding promoter signals were not recognized in Mycobacterium smegmatis, in whichtranscription of lhplesat-6 is initiated at different locations. The M. tuberculosis lhp gene product was identified as CFP-10, a low- molecular-mass protein found in the short-term culture filtrate. These results show that the genes encoding CFP-10 and ESAT-6 are transcribed together in M. tuberculosis and that both code for small exported proteins.
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Isolation of carbon- and nitrogen-deprivation-induced loci of Sinorhizobium meliloti 1021 by Tn5-luxAB mutagenesis
More LessSUMMARY: Soil bacteria, such as Sinorhizobium meliloti, are subject to variation in environmental conditions, including carbon- and nitrogen-deprivation. The ability of bacteria to sense changes in their environment and respond accordingly is of vital importance to their survival and persistence in the soil and rhizosphere. A derivative of Tn5 which creates transcriptional fusions to the promoterless luxAB genes was used to mutagenize 5. meliloti 1021 and 5000 insertion mutants were subsequently screened for gene fusions induced by selected environmental stresses. The isolation of 21 gene fusions induced by nitrogen-deprivation and 12 induced by carbon-deprivation is described. Cloning and partial DNA sequence analysis of the transposon-tagged loci revealed a variety of novel genes, as well as S. meliloti genes with significant similarity to known bacterial loci. In addition, nodule occupancy studies were carried out with selected TnSluxAB insertion mutants to examine the role of the tagged genes in competition.
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Functional characterization of the Erwinia chrysanthemi Outs protein, an element of a type II secretion system
More LessSUMMARY: Secretion of pectate lyases and a cellulase occurs in winis chrysanthemi through a type II secretion machinery, the Out system. Proper insertion of the secretin OutD in the outer membrane requires the presence of Outs. Outs is an outer-membrane lipoprotein that interacts directly with OutD. Using ligand-blotting experiments, it has been shown that this interaction requires at least the 62 C-terminal amino acids of OutD. When this domain was added to the C-terminal extremity of the secreted pectate lyase PelD, the construct was stabilized by Outs but not inserted into the outer membrane. Thus, this domain is sufficient to interact with Outs but it is unable to confer the ability to be inserted into the outer membrane in the presence of Outs. A screen for outs mutants unable to secrete pectate lyases gave only mutants unable to properly localize OutD in the outer membrane and no mutant in the protection function. Thus, the interaction between Outs and OutD can probably not be abolished by the mutation of a single amino acid, and the insertion of OutD in the outer membrane may require additional proteins.
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Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate
More LessSUMMARY: Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligems NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced M, of 6OOOO, consistent with the observed M, (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 molO/O), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon sorce was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned 5. clavuligerus aceB gene was expressed in Escherichia coli BL2l (DE3).Transformants exhibited an approximately 713old increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.
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- Genomics
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Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae
More LessSUMMARY: The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia colichromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the o her end. The murD, murG and diw/B genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivlVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.
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Construction of a physical and preliminary genetic map of Aerornonas hydrophila IMP636
More LessSUMMARY: A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed. The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by 51 nuclease. Cleavage of the chromosome with Pacl and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb. Digestion of the chromosome with I-Ceul produced 10 fragments, indicating that 10 rrl(23S) genes were likely t o be present. Hybridizations between DNA fragments generated with Pad, PmeI and I-Ceul were used t o initially determine the relationship between these segments. To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for Pad and PmeI, producing pJP9540. Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, Pad and Pmel cleavage determined the location of genes within macrorestriction fragments with the additional bands produced forming hybridization probes. From the data generated, it was possible t o form a physical map comprising all the fragments produced by Pacl and Pmel, and assign the contig of I-Ceul fragments on this map. The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data.
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A vector for systematic gene inactivation in Bacillus subtilis
More LessSUMMARY: To study the functions of the uncharacterized open reading frames identified in the Bacillus subtih genome, several vectors were constructed t o perform insertional mutagenesis in the chromosome. All the pMUTlN plasmids carry a lac2 reporter gene and an inducible Pspac promoter, which is tightly regulated and tan be induced about 1000-fold. The integration of a pMUTlN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lac2 becomes transcriptionally fused t o the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subti/is genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTlN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTlN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.
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The product of the yvoC(geri)gene of Bacillus subtillis is required for spore germination
More LessSUMMARY: All known gerF mutations affecting Bacillus subtilis spore germination have been mapped, by a combination of recombination and complementation analysis, to yvoC (/gt)# a gene belonging to the yvoB CptsK) yvoC (/gt) yvoDEF operon. Examination of the properties of null mutants confirmed that the only gene in the operon that affects germination is poC, which encodes a homologue of known prelipoprotein diacylglyceryl transferases. As several germination proteins (GerAC, GerBC, GerKC, GerD) are predicted lipoproteins, it is not unreasonable to assume that a defect in prelipoprotein processing will affect spore germination. Two other null mutants in this chromosomal region showed a clear phenotype: the nagA gene is required for growth on N-acetylglucosamine, whereas a null mutation in yvoB (ptsK) affects colony formation from single cells.
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- Pathogenicity And Medical Microbiology
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Internalization and cytotoxicity are important virulence mechanisms in vibrio-f ishepithelial cell interactions
More LessSUMMARY: Vibrio anguillarum and Vibrio damselee are Gram-negative bacteria that cause systemic infections called vibriosis in fish. They can enter fish cells and survive as intracellular parasites. The host-pathogen interactions between these Vibrio species and the fish epithelial cell lines epithelioma papillosum of carp (EPC) and grunt-f in tissue (GF) cells, were examined using phase-contrast, scanning electron and confocal microscopy. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Some Vibrio species induced morphological changes in fish cells and this allowed classification into a cytopathic group and a noncytopathic group. The cytopathic group could be subdivided into two invasive groups (I and II) and a cytotoxic group. Of the invasive strains V. anguillarum 811218-5W (group I) and GNirus/5(3) (group II), genistein, a tyrosine kinase inhibitor, only inhibited internalization of V. anguillarum GNirus/5(3) into EPC cells, whereas staurosporine, a protein kinase C inhibitor, accelerated internalization of both strains. Cytochalasin D, an inhibitor of microfilament polymerization, prevented internalization of both strains, whilst vincristin, a microtubule inhibitor, only inhibited internalization of V. anguillarum GNirus/5(3). For the cytotoxic strain V. damselae ATCC 33539, extracellular products (ECP) alone caused morphological changes in EPC and GF. Bacterial internalization may not be important in the pathogenesis of this group. The non-cytopathic strain V. anguillarum SU5/93(2) did not enter cells or induce any changes in EPC and GF monolayen. This study has identified some major differences between Vibrio species in their interactions with fish cells in vitm and will thus facilitate future studies of the molecular basis of pathogenesis of vibriosis.
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Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes
SUMMARY: Entembacter aemgenes is among the five most frequently isolated nosocomial pathogens in France, and this bacterium also shows increasing multidrug resistance. In this study, various E. aerogenes strains isolated from hospital units were characterized for their outer-membrane proteins, antibiotic susceptibilities (inhibition diameters and MICs) and resistance mechanisms associated with modification of envelope permeability (porin alteration and active efflux). Diminished outer-membrane permeability due to porin alterations was found in conjunction with the expression of an enzymic barrier in resistant isolates. Interestingly, changes in the functional expression of porins appeared to play a special role in susceptibility to cefepime. An active efflux to quinolones was also identified. Simultaneous changes in envelope permeability, i.e. a porin deficiency (in) and an efflux mechanism (out), were clearly evident in two clinical strains.
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Characterization of Trichornonas vaginalis AP33 adhesin and cell surface interactive domains
More LessSUMMARY: Adherence to host target cells is a critical step in establishing infection with the sexually transmitted pathogen Tiichomonas vaginalis. Four parasite surface proteins mediating attachment to vaginal epithelial cells have been identified. One surface protein, termed AP33, was characterized further to identify domains interactive with previously generated antibodies and with host surface sites. N- and C-terminal deletion subclones were generated and tested for reactivity with both mAb and rabbit antiserum against AP33, and were also examined for their ability to bind to host cells. Surprisingly, the rabbit antiserum known to inhibit cytoadherence recognized an epitope(s) contained within 72 residues in the N-terminal half of the protein. However, the mAb epitope was immunoreactive with a 28-amino-acid region near the C-terminus. Subsequent mapping of this region with overlapping peptides identified a nine-amino-acid sequence reactive with the mAb. Equally surprising, two domains interactive with host cell surfaces were identified at distinct parts of AP33: one in the N-terminal half of the protein, and the other within 24 residues in the C-terminal third. Further analysis of the C-terminal binding domain revealed that a peptide representing this area could inhibit T. vaginalis cytoadherence by 40%.
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A lipid A-associated protein of Porphyromonas gingivalis, derived from the haemagglutinating domain of the RI protease gene family, is a potent stimulator of interleukin 6 synthesis
SUMMARY: There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivdis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81,68,48,47,28,25,20,17 and 16 kDa. These LAPS, at concentrations as low as 100 ng mV, were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood t o produce the pro-inflammatory cytokine IL-6. The cytokine-inducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous t o an internal region of a conserved adhesin domain contained within a family of P. gingivdis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivelis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear t o have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influencethe cytokine status of the periodontiurn in patients with the common chronic inflammatory conditions known as the periodontal diseases.
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A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease
More LessSUMMARY: The population biology of Streptococcus pneumoniae is poorly understood. Most of the important issues could be addressed by the molecular characterization of large, well sampled populations from carriage and from the different manifestations of pneumococcal disease. The authors have therefore developed a pneumococcal multilocus sequence typing scheme and database by sequencing - 450 bp fragments of seven housekeeping loci from 295 isolates. The combination of alleles at the seven loci provided an allelic profile, or sequence type (ST), and the relatedness between isolates was obtained by constructing a dendrogram from the matrix of paiwvise differences between STs. The typing scheme was validated using pneumococci of known genetic relatedness and could resolve 6 billion STs. Among 274 isolates from recent cases of invasive pneumococcal disease in eight countries,143 STs were resolved, but 12 STs contained at least five isolates (range 5-21 isolates). The repeated recovery of indistinguishable isolates from invasive disease in different countries implies that these STs define strains with an increased capacity to cause invasive disease. The relationship between STs and serotypes suggested that, in the longer term, capsular genes have been distributed horizontally within the pneumococcal population, but in the short term, expansion of clones occurs with only occasional changes of serotype. The multilocus sequence typing scheme provides a powerful new approach to the characterization of pneumococci, since it provides molecular typing datathat are electronically portable between laboratories, and which can be used to probe aspects of the population and evolutionary biology of these organisms. A Web sitefor themolecular characterization of pneumococci by MLST is available (http://mlst.zoo.ox.ac.uk).
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Siderotyping of fluorescent pseudomonads:characterization of pyoverdines of Pseudornonas fluorescens and Pseudornonas putida strains from Antarctica
SUMMARY: Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens lOcW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour t o each other, whereas the fifth strain, P. fluorescens 5lW, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 1OcW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared t o be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.
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- Physiology And Growth
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PhhC is an essential aminotransferase for aromatic amino acid catabolism in Pseudornonas aeruginosa
More LessSUMMARY: The phhC gene of Pseudomonas aeruginosa encodes a protein which is a member of the Family I aminotransferases. At high expression levels in the heterologous Escherichia oli system, PhhC can compensate for the absence of AspC (which functions in L-aspartate biosynthesis) and TyrB (which functions in aromatic biosynthesis). In the native organism, PhhC is essential for catabolism of either L-tyrosine or L-phenylalanine, as demonstrated by gene inactivation. This catabolic function of PhhC is consistent with its inclusion as the distal gene in the inducible phenylalanine hydroxylase operon. The presence of PhhC for catabolism of aromatic amino acids is required in spite of an existing multiplicity of other P. aeruginosa aminotransferases having a similar pattern of broad substrate specificity in vitm. This implies a spatial orientation of PhhC that effectively specializes it for aromatic amino acid catabolism.
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Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF, are induced by extracellular pyochelin in Pseudornonas aeruginosa
More LessSUMMARY: The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pChEF8 encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE(156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensationkyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchf-'/acZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led t o Fur (ferric uptake regulator)-dependent repression of the pchE -laciZ fusion. A translational pchD-lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.
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Molecular and functional analysis of pTAV320, a repABC-type replicon of the Paracoccus versutus composite plasmid pTAV1
More LessSUMMARY: The second replicator region of the native plasmid pTAVl of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (43 kb) was cloned and sequenced. pTAV320 encodes three putative proteins - RepA, RepB and RepC. This replicator region shows strong structural and functional similarity to mpABC-type rep1 icons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown t o be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a rninireplicon derived from the other pTAVl replicator region. Deletions introduced into the mpC gene abolished the ‘stabilizing’ activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. wersutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodohcter strains in addition t o P. versutus.
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Isolation and characterization of Staphylococcus aureus starvat ion4 nduced, stationary-phase mutants defective in survival or recovery
More LessSUMMARY: Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTVI transposon insertion libraries and were designated suv as they had apparent-suryival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suw-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the 5. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LNI insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in theidentification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-I), haem A synthase (suv-3)# a component of the 505 response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTVI insertion created lac2 transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-II were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.
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Streptococcus pyogenes protein F promotes invasion of HeLa cells
More LessSUMMARY: Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using 111916 insertion mutagenesis or an isogenic 5. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with 5. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did notpermit internalization of the E. coli cells.
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