- Volume 147, Issue 10, 2001
Volume 147, Issue 10, 2001
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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Porins from Salmonella enterica serovar Typhimurium induce TNF-α, IL-6 and IL-8 release by CD14-independent and CD11a/CD18-dependent mechanisms
Lipopolysaccharide (LPS) of Gram-negative bacteria and several surface components of Gram-positive bacteria utilize CD14 and CD11a/18 as cellular receptors to induce expression and release of cytokines. Of the surface components of Gram-negative bacteria, porins exhibit a biological activity similar to that of LPS. The results in this paper show that the mechanism of stimulation by porins of THP-1 cells enriched in CD14 receptor after treatment with 1,25-dihydroxyvitamin D3 (vitamin D3) is independent of this receptor, but is partially dependent on CD11a/18 integrins.
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- Biochemistry
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Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina
More LessThe biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts. The NAD+:isocitrate dehydrogenases of Mc. circinelloides and Mort. alpina were not absolutely dependent on AMP for activity. Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts. In Mc. circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool. Pyruvate carboxylase in Mc. circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi. As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi.
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- Bioenergetics And Transport
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Cytochrome c oxidase contains an extra charged amino acid cluster in a new type of respiratory chain in the amino-acid-producing Gram-positive bacterium Corynebacterium glutamicum
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB052748 and AB052749.
The membranes from Corynebacterium glutamicum cells contain a hydrophobic di-haem C protein as the cytochrome c subunit of the new type of cytochrome bc complex (complex III in the respiratory chain) encoded by the qcrCAB operon [Sone, N., Nagata, K., Kojima, H., Tajima, J., Kodera, Y., Kanamaru, T., Noguchi, S. & Sakamoto, J. (2001). Biochim Biophys Acta 1503, 279–290]. To characterize complex IV, cytochrome c oxidase and its structural genes were isolated. The oxidase is of the cytochrome aa 3 type, but mass spectrometry indicated that the haem is haem As, which contains a geranylgeranyl side-chain instead of a farnesyl group. The enzyme is a SoxM-type haem–copper oxidase composed of three subunits. Edman degradation and mass spectrometry suggested that the N-terminal signal sequence of subunit II is cleaved and that the new N-terminal cysteine residue is diacylglycerated, while neither subunit I nor subunit III is significantly modified. The genes for subunits II (ctaC) and III (ctaE) are located upstream of the qcrCAB operon, while that for subunit I (ctaD) is located separately. The oxidase showed low enzyme activity with extrinsic substrates such as cytochromes c from horse heart or yeast, and has the CuA-binding motif in its subunit II. A prominent structural feature is the insertion of an extra charged amino acid cluster between the β2 and β4 strands in the substrate-binding domain of subunit II. The β2–β4 loop of this oxidase is about 30 residues longer than that of major cytochrome c oxidases from mitochondria and proteobacteria, and is rich in both acidic and basic residues. These findings suggest that the extra charged cluster may play a role in the interaction of the oxidase with the cytochrome c subunit of the new type of bc complex.
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The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines
More LessThe active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important antibiotics. Cells expressing LmrP display an increased resistance to the lincosamide, streptogramin, tetracycline and 14- and 15-membered macrolide antibiotics. The streptogramin antibiotic quinupristin, present in the fourth-generation antibiotic RP 59500, can inhibit LmrP-mediated Hoechst 33342 transport, but is not transported by LmrP, indicating that quinupristin acts as a modulator of LmrP activity. LmrP-expressing Lactococcus lactis cells in which a proton-motive force is generated accumulate significantly less tetracycline than control cells without LmrP expression. In contrast, LmrP-expressing and control cells accumulate equal amounts of tetracycline in the absence of metabolic energy. These findings demonstrate that the increased antibiotic resistance in LmrP-expressing cells is a result of the active extrusion of antibiotics from the cell.
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- Environmental Microbiology
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Fluoranthene metabolism in Mycobacterium sp. strain KR20: identity of pathway intermediates during degradation and growth
More LessMycobacterium sp. strain KR20, which was isolated from a polycyclic aromatic hydrocarbon (PAH) contaminated soil of a former gaswork plant site, metabolized about 60% of the fluoranthene added (0·5 mg ml−1) to batch cultures in mineral salts medium within 10 d at 20 °C. It thereby increased its cell number about 30-fold and produced at least seven metabolites. Five metabolites, namely cis-2,3-fluoranthene dihydrodiol, Z-9-carboxymethylene-fluorene-1-carboxylic acid, cis-1,9a-dihydroxy-1-hydro-fluorene-9-one-8-carboxylic acid, 4-hydroxybenzochromene-6-one-7-carboxylic acid and benzene-1,2,3-tricarboxylic acid, could be identified by NMR and MS spectroscopic techniques and ascribed to an alternative fluoranthene degradation pathway. Besides fluoranthene, the isolate could not use any of the PAHs tested as a sole source of carbon and energy.
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- Genetics And Molecular Biology
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The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol
More LessPseudomonas aeruginosa ATCC 17933 uses a pyrroloquinoline quinone-dependent ethanol oxidation system. Two mutants of P. aeruginosa, unable to grow on ethanol and showing no acetyl-CoA synthetase (ACS) activity under standard test conditions, were complemented by cosmid pTB3018. Subcloning led to the isolation of a gene which encodes a protein with high similarity to acetyl-CoA synthetases. Interruption of the putative acsA gene by a kanamycin-resistance cassette resulted in a mutant also unable to grow on ethanol and with very low residual acetyl-CoA-forming activity. Complementation by the wild-type allele of the acsA gene restored growth and led to the expression of ACS activity in excess of that of wild-type cells. In wild-type P. aeruginosa, ACS activity was induced upon growth on ethanol, 2,3-butanediol, malonate and acetate. The wild-type and mutants defective in ACS activity showed an active acetate kinase (ACK) under the growth conditions used; however, phosphotransacetylase (PTA) could not be detected. The data indicate that P. aeruginosa requires active acsA gene product for growth on ethanol.
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Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes
More LessListeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin. Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain. The two proteins were efficiently secreted into the culture supernatant of L. monocytogenes and were able to bind to cell membranes. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.
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Genomic analysis and growth-phase-dependent regulation of the SEF14 fimbriae of Salmonella enterica serovar Enteritidis
More LessThe GenBank accession number for the sequence reported in this paper is AF239978.
Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.
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Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile
More LessThe GenBank accession number for the Tn5398 element and flanking sequence is AF109075.
Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolide-lincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.
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Differentiation of Campylobacter species by AFLP fingerprinting
The fluorescent amplified fragment length polymorphism (AFLP) fingerprinting method was tested for its ability to identify and subtype the most important Campylobacter species found in veterinary infections. Sixty-nine reference strains and 19 clinical isolates of Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis, Campylobacter coli, Campylobacter lari, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, Campylobacter mucosalis, Campylobacter helveticus and Campylobacter sputorum were subjected to analysis. The topology of the dendrogram obtained by numerical analysis of the AFLP profiles did not reflect the phylogenetic relationships as derived from 16S rDNA sequence comparison. However, except for C. lari, AFLP analysis grouped the strains that belonged to the same genomic species into distinct clusters. C. lari strains were separated into two distinct AFLP groups, which corresponded with nalidixic-acid-sensitive and -resistant variants of C. lari. These results correlated with data from whole-cell protein profiling. Within C. jejuni, C. hyointestinalis and C. fetus, strains could be identified at the subspecies level. AFLP analysis also allowed the subtyping of most species at the strain level. It is concluded that AFLP analysis is a valuable tool for concurrent identification of campylobacters at the species, subspecies and strain levels. In addition, the data confirm and extend previous reports showing that C. lari is a heterogeneous species that may comprise multiple taxa.
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The replicon of pSW800 from Pantoea stewartii
More LessThe GenBank accession number for the sequence of the minimal replicon of pSW800 is AF310258.
A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes – repA and repB, which encode a 36·5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem–loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem–loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5′-GCACGGG-3′) located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem–loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIα and IncL/M groups also regulates pSW800 replication.
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The transcriptional activator NhaR is responsible for the osmotic induction of osmC p1, a promoter of the stress-inducible gene osmC in Escherichia coli
More LessTwo overlapping promoters, osmC p1 and osmC p2, direct the transcription of the osmC gene of Escherichia coli. The proximal promoter, osmC p2, is induced upon entry into stationary phase under the control of Eσs, the RNA polymerase that uses the σs (RpoS) sigma factor. Transcription from the distal promoter, osmC p1, is independent of σs. Previous analysis demonstrated that the osmolarity of the growth medium modulates expression of both promoters. The use of an E. coli genomic library showed that the cloned nhaR gene was able to stimulate transcription of an osmC–lac reporter fusion. NhaR is a positive regulator of the LysR family, previously identified as an activator of nhaA, a gene encoding a Na+/H+ antiporter involved in adaptation to Na+ and alkaline pH in E. coli and other enteric bacteria. NhaR was shown to activate only the expression of osmC p1 and to be necessary for the induction of this promoter by LiCl, NaCl and sucrose. Therefore, activation by NhaR is responsible for the osmotic induction of osmC p1. In contrast to its action on nhaA, NhaR activation of osmC p1 is independent of H-NS. Activation of osmC p1 by NhaR requires a site located just upstream of the atypical −35 region of the promoter.
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Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron
This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague.
The GenBank accession number for the sequence reported in this paper is AF130307.
The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46·5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46·5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.
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The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene
The GenBank accession number for the sequence reported in this paper is AF262220.
J He, N Magarvey, M Piraee and L. C ViningRegions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7·5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7·5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.
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The Chryseobacterium meningosepticum PafA enzyme: prototype of a new enzyme family of prokaryotic phosphate-irrepressible alkaline phosphatases?
The GenBank accession number for pafA reported in this paper is AF157621.
Chryseobacterium meningosepticum is an aerobic Gram-negative rod widely distributed in natural environments. Unlike many bacteria, it produces a phosphate-irrepressible periplasmic alkaline phosphatase (AP). This work describes cloning of the gene encoding that enzyme from C. meningosepticum CCUG 4310 (NCTC 10585), and preliminary characterization of its product. The gene, named pafA, encodes a protein (PafA) of 546 amino acids with a calculated molecular mass of the mature peptide of 58682 Da. PafA exhibits high sequence identity with the PhoV AP of Synechococcus PCC 7942 (49·9% identity) and with the Cda Ca2+-dependent ATPase of Myroides odoratus (51·9% identity), while being more distantly related to the PhoD AP of Zymomonas mobilis (22·1% identity) and to the PhoA AP of Escherichia coli (14·0% identity). PafA was partially purified; it exhibits optimal activity at pH 8·5 and is active towards a broad spectrum of substrates including both phosphomonoesters and ATP, with preferential activity for the latter compound. The present findings allow definition of a new family of APs including 60 kDa, periplasmic enzymes whose expression is not influenced by freely available Pi in the medium. Moreover, PafA can be considered an evolutionary intermediate between Ca2+-ATPase of M. odoratus and the APs PhoV of Synechococcus PCC 7942 and PhoD of Z. mobilis.
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Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate
More LessStreptococcus mutans produces a number of extracellular sucrose-metabolizing enzymes that contribute to the ability of the organism to cause dental caries, including three glucosyltransferases, the products of the gtfB, gtfC and gtfD genes, and a fructosyltransferase, encoded by the ftf gene. To better understand the regulation of the expression of these genes under environmental conditions that more closely mimic those in dental plaque, two strains of S. mutans harbouring fusions of the gtfBC (SMS102) and ftf (SMS101) promoters to a chloramphenicol acetyltransferase (CAT) gene were examined in biofilms formed in vitro. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium. CAT specific activity in biofilm cells was measured at quasi-steady state or following additions of 25 mM sucrose or glucose, with or without pH control. After approximately 10 generations of biofilm growth, the ftf and gtfBC genes of S. mutans were found to be expressed at levels different from those reported for planktonic cells growing under otherwise similar conditions. The expression of these genes was induced by the addition of sucrose to the quasi-steady-state cultures. Expression of the gtfBC genes was influenced by environmental pH, since CAT specific activities in quasi-steady-state biofilms of strain SMS102 grown without pH control were twice those produced by cells grown with pH control. Moreover, addition of glucose to quasi-steady-state biofilms resulted in increased expression of the gtfBC–cat fusion, although the magnitude of the induction was less than that seen with sucrose. The effect of pH on ftf expression was negligible. A modest and transient induction of ftf was observed in biofilms pulsed with excess glucose and the kinetics and level of induction of ftf by excess carbohydrate were dependent on the pH of the biofilms. This study demonstrates that the type and amount of carbohydrate and the environmental pH have a major influence on transcription of the gtfBC and ftf genes when the organisms are growing in biofilms, and provides evidence for previously undisclosed regulatory circuits for exopolysaccharide gene expression in S. mutans.
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- Genomics
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FindTarget: software for subtractive genome analysis
More LessIn silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. By this method, one searches for genes present in one group of bacteria and absent in another group. A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis. The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome. The searches are based on BLASTP comparisons of proteomes. FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results. To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented. The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.
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- Pathogenicity And Medical Microbiology
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Adherence of Burkholderia cepacia to respiratory tract epithelial cells and inhibition with dextrans
More LessAdherence of Burkholderia cepacia to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date, no effective anti-adhesive strategy has been devised for preventing B. cepacia infection in CF patients. It was found in this study that B. cepacia adhered to respiratory epithelial cells both in vitro and in vivo. However, strains with cable-like pili (Cbl) exhibited the typical clump formation on pneumocytes, whereas non-cable piliated strains predominantly showed single-cell adherence. Dextrans (nominally 4000–10000 Da) significantly inhibited adhesion of B. cepacia to A549 pneumocytes. When compared on an equal weight basis, the nominally 10000 Da dextran was most inhibitory. A dose-dependent inhibitory effect (up to 80 mg ml−1) was observed for most strains. Dextran exerted less of an anti-adhesive effect on the two Cbl+ strains than on the others which were Cbl−. Dextrans appeared to block the adherence in a non-specific fashion, as shown by the observations that the inhibitory effect was readily reversible and oligosaccharides composed of 2–4 glucose units with the same α-1,6 linkage were not inhibitory. The mean molecular masses of dextrans used in this study, as determined by gel filtration and MS, were approximately 10-fold lower than those indicated by the manufacturers. Our data suggest that dextran of nominal molecular mass 4000 Da at a concentration of 40 mg ml−1 (10 mM according to manufacturer’s quoted molecular mass) or more may be useful in patients with CF to prevent colonization and infection with B. cepacia.
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Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa
More LessThe type III secretion system of Pseudomonas aeruginosa transports four known effector proteins: ExoS, ExoT, ExoU and ExoY. However, the prevalence of the type III secretion system genes or the effector-encoding genes in clinical and environmental isolates of P. aeruginosa has not been well studied. Southern hybridization analyses and PCR were performed on over 100 P. aeruginosa isolates to determine the distribution of these genes. Clinical isolates were obtained from urine, endotracheal, blood and wound specimens, from the sputum of cystic fibrosis (CF) patients, and from non-hospital environmental sites. The popB gene was used as a marker for the presence of the large chromosomal locus encoding the type III secretion machinery proteins. Each isolate contained the popB gene, indicating that at least a portion of this large chromosomal locus was present in all isolates. Likewise, each isolate contained exoT-like sequences. In contrast, the exoS, exoU and exoY genes were variable traits. Overall, 72% of examined isolates contained the exoS gene, 28% contained the exoU gene, and 89% contained the exoY gene. Interestingly, an inverse correlation was noted between the presence of the exoS and exoU genes in that all isolates except two contained either exoS or exoU but not both. No significant difference in exoS, exoU or exoY prevalence was observed between clinical and environmental isolates or between isolates cultured from different disease sites except for CF respiratory isolates. CF isolates harboured the exoU gene less frequently and the exoS gene more frequently than did isolates from some of the other sites of infection, including the respiratory tract of patients without CF. These results suggest that the P. aeruginosa type III secretion system is present in nearly all clinical and environmental isolates but that individual isolates and populations of isolates from distinct disease sites differ in their effector genotypes. The ubiquity of type III secretion genes in clinical isolates is consistent with an important role for this system in human disease.
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- Physiology And Growth
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Relationship between nucleic acid ratios and growth in Listeria monocytogenes
More LessListeria monocytogenes is a pathogen whose distribution in a range of foodstuffs requires the development of methods for sensitive and rapid detection. Molecular biological methods usually rely on specific detection of L. monocytogenes rDNA directly amplified by the application of PCR to DNA extracts. Information on the metabolic status of L. monocytogenes populations would be valuable and can, in theory, be provided by quantitative detection of rRNA itself. Both fluorometry and oligonucleotide probe assays were applied to L. monocytogenes cultures to quantify RNA and DNA and produced more meaningful data than previous estimates for bacteria based on eukaryotic nucleic acid standards. In batch culture, the RNA–DNA ratio was found to be greatest at the end of exponential growth, after which RNA became degraded in accordance with the rapid decrease in viability. When the pH of the medium was controlled at neutrality, culture viability was dramatically extended and although RNA was degraded, intact DNA was maintained for the duration of the experiment. Ribosome numbers per cell were estimated to decrease from about 25000 observed during mid-exponential growth to about 600 during stationary phase, under pH-controlled conditions. Like Escherichia coli, therefore, L. monocytogenes loses viability and rRNA rapidly once exponential growth has ceased in batch culture. However, much improved survival of a culturable L. monocytogenes population when pH is controlled has clear implications for the persistence of this species in buffered environments such as dairy products.
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Fermentable-sugar-level-dependent regulation of leukotoxin synthesis in a variably toxic strain of Actinobacillus actinomycetemcomitans
More LessThe GenBank/EMBL/DDBJ accession number for the sequence data in this paper is AB054839.
Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells)−1 to 3·1 pmol (mg dry wt cells)−1, which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.
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Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds
Polycations [protamine, polymyxin B nonapeptide (PMBN) and polyethyleneimine (PEI)] have been shown to increase the cell wall permeability of Mycobacterium vaccae to highly hydrophobic compounds, as manifested in enhanced intracellular bioconversion of β-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD), and cell sensitization to erythromycin and rifampicin. The quantity of AD(D) formed per biomass unit was twice as high in the presence of PMBN and PEI, and three times higher with protamine. The sensitization factor, i.e. the MIC50 ratio of the control bacteria to those exposed to polycations, ranged from 4 to 16, depending on the polycation/antibiotic combination. Non-covalently bound free lipids were extracted from the control and polycation-treated cells and fractionated with the use of chloroform, acetone and methanol. Chloroform- and acetone-eluted fractions (mainly neutral lipids and glycolipids, respectively) showed significant polycation-induced alterations in their quantitative and qualitative composition. The fatty acid profile of neutral lipids was reduced in comparison to control, whereas acetone-derived lipids were characterized by a much higher level of octadecenoic acid (C18:1) and a considerably lower content of docosanoic acid (C22:0), the marker compound of mycolate-containing glycolipids. Methanol-eluted fractions remained unaltered. Cell-wall-linked mycolates obtained from delipidated cells were apparently unaffected by the action of polycations, as judged from the TLC pattern of mycolic acid subclasses, the mean weight of mycolate preparations and the C22:0 acid content in the mycolates, determined by GC/MS and pyrolysis GC. The results suggest the involvement of the components of non-covalently bound lipids in the outer layer in the M. vaccae permeability barrier.
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New aspects of the glucose activation of the H+-ATPase in the yeast Saccharomyces cerevisiae
More LessThe glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983 R27 . Many aspects of this signal transduction pathway are still obscure. In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process. It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase. The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated. Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed. Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H+-ATPase.
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- Systematics And Evolution
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Genetic diversity of Pasteurella multocida fowl cholera isolates as demonstrated by ribotyping and 16S rRNA and partial atpD sequence comparisons
More LessThe GenBank accession numbers for the 16S rRNA sequences of strain HIM 830-7T (NCTC 10204T) and 77179 of P. multocida subsp. gallicida and HIM 746-6T (NCTC 11619T) of P. multocida subsp. septica are AF326323, AF326324 and AF326325, respectively; those for the atpD nucleotide sequences are in Fig. 3.
The genetic diversity of Pasteurella multocida, the aetiological agent of fowl cholera, was investigated. The strain collection comprised 69 clinical isolates representing a wide spectrum of hosts and geographic origin. The three type strains for the subspecies of P. multocida were also included. Avian isolates of P. multocida subsp. multocida and P. multocida subsp. septica did not represent separate lines by HpaII ribotyping and the two type strains of mammalian origin (porcine and cat bite) seemed to be representative of avian strains of P. multocida subspp. multocida and septica. By ribotyping, all P. multocida subsp. gallicida strains, except one chicken isolate and the type strain, clustered together. This indicated that the bovine type strain was not representative of this subspecies and that most strains of P. multocida subsp. gallicida are genetically related and may be distantly related to other P. multocida isolates, including those of avian origin. By 16S rRNA and atpD sequence comparisons of selected strains, including both P. multocida isolated from birds and mammals and selected distantly related Pasteurella species associated with birds and mammals, it was found that P. multocida is monophyletic. Extended DNA–DNA hybridizations are highly indicated since strains may exist which would connect the existing subspecies at species level. The considerable genetic diversity of P. multocida fowl cholera isolates is probably related to the clonal nature of this organism, resulting in many divergent lines.
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