- Volume 148, Issue 11, 2002
Volume 148, Issue 11, 2002
- Review Article
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- Microbiology Comment
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- Research Paper
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Bovicin HC5, a bacteriocin from Streptococcus bovis HC5
More LessPrevious work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and α-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2·0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.
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Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes b
More LessbThe GenBank accession numbers for the sequences reported in this paper are given in Methods.
Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic–aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-β-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1·8% P (sludge Q) and 1·5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99·7% identical, forming a cluster in the γ-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the γ-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the γ-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel γ-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the γ-Proteobacteria radiation be named ‘Candidatus Competibacter phosphatis’.
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Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans
More LessAntibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like ’stringent’ RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.
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Characterization of mutations in aclacinomycin A-non-producing Streptomyces galilaeus strains with altered glycosylation patterns
In this study a set of Streptomyces galilaeus ATCC 31615 mutants was characterized, which are incapable of synthesizing some or all of the deoxyhexose sugars of aclacinomycin A. Complementation experiments with the the mutant strains H026, H038, H039, H054, H063, H065 and H075 were carried out with glycosylation genes previously derived from the wild-type S. galilaeus. Mutations in strains H038, H063 and H075 were complemented with single PCR-amplified genes. Furthermore, amplification and sequencing of the corresponding genes from the mutant strains revealed single point mutations in the sequences. First, in H038 a transition mutation in aknQ, encoding a putative dTDP-hexose 3-ketoreductase, causes an amino acid substitution from glycine to aspartate, suppressing the biosynthesis of both 2-deoxyfucose and rhodinose and thus leading to the accumulation of aclacinomycin T with rhodosamine as its only sugar. Second, in H063, which accumulates aklavinone without a sugar moiety, amino acid substitution occurs, with threonine being substituted by isoleucine in dTDP-glucose synthase, the first enzyme participating in deoxyhexose biosynthesis, encoded by aknY. Third, a nonsense mutation in aknP leads to truncated dTDP-hexose 3-dehydratase in H075, which is incapable of synthesizing rhodinose. In addition, mutants H054 and H065, which accumulate aclacinomycins without aminosugars, were complemented by a gene for an aminotransferase, aknZ. Characterization of the nature of the mutations adds to the usefulness and value of the mutants in the analysis of gene function and in the creation of novel compounds by combinatorial biosynthesis. Furthermore, these results strengthen the assignments of akn gene products and enlighten the biosynthetic pathway for deoxyhexoses.
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Streptococcus mutans biofilm formation: utilization of a gtfB promoter–green fluorescent protein (PgtfB::gfp) construct to monitor development
More LessThe glucosyltransferases of Streptococcus mutans are recognized as important virulence factors for this cariogenic bacterium. To study the expression of the gtfB gene of S. mutans in biofilms, a gtfB promoter (PgtfB)–green fluorescent protein (GFP) reporter system was developed. A Streptococcus–Escherichia coli shuttle vector harbouring a PgtfB::gfp cassette was introduced into S. mutans GS-5, and the expression of GFP by the transformed S. mutans cells was confirmed by fluorescence microscopy. Furthermore, confocal laser scanning microscopy was carried out on biofilms attached to polystyrene plates; enhanced gtfB expression was observed in various microcolonies across these biofilms. To further test the hypothesis that gtfB expression is upregulated in biofilms, flow cytometry analysis was done on planktonic and biofilm cells; this analysis showed an approximately five-fold increase in gtfB expression in the biofilm cells relative to the planktonic cells. Real-time (TaqMan) PCR analysis confirmed that gtfB expression in the biofilm cells was enhanced relative to the planktonic cells. Previously, it has been suggested that the S. mutans gtfC gene might be co-transcribed with gtfB. Therefore, RT-PCR analysis was performed on gtfB-expressing S. mutans; this analysis demonstrated that gtfC was co-transcribed with gtfB. These results indicated that GFP expression can be utilized to examine gene regulation in S. mutans biofilm formation.
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Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins
Native and recombinant FomA proteins were extracted by detergent from the cell envelopes of Fusobacterium nucleatum and Escherichia coli, and purified to near homogeneity by chromatography. Circular dichroism analysis revealed that the FomA protein consists predominantly of β-sheets, in line with the previously proposed 16-stranded β-barrel topology model. Results obtained by trypsin treatment of intact cells and cell envelopes of F. nucleatum, and from limited proteolysis of purified FomA protein, indicated that the N-terminal part of the FomA protein is not an integral part of the β-barrel, but forms a periplasmic domain. Based on these results a new topology model is proposed for the FomA protein, where the C-terminal part forms a 14-stranded β-barrel separate from the periplasmic N-terminal domain.
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Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM
More LessBacillus subtilis 168 was assayed for its growth on tricarboxylic acid (TCA) cycle intermediates and related compounds as the sole carbon sources. Growth of the organism was supported by citrate, D-isocitrate, succinate, fumarate and L-malate, whereas no growth was observed in the presence of cis-aconitate,2-oxoglutarate, D-malate, oxaloacetate and tricarballylate. Growth of the organism on the tricarboxylates citrate and D-isocitrate required the presence of functional CitM, an Mg2+–citrate transporter, whereas its growth on succinate, fumarate and L-malate appeared to be CitM-independent. Interestingly, the naturally occurring enantiomer D-isocitrate was favoured over L-isocitrate by the organism. Like citrate, D-isocitrate was shown to be an inducer of citM expression in B. subtilis. The addition of 1 mM Mg2+ to the growth medium improved growth of the organism on both citrate and D-isocitrate, suggesting that D-isocitrate was taken up by CitM in complex with divalent metal ions. Subsequently, the ability of CitM to transport D-isocitrate was demonstrated by competition experiments and by heterologous exchange in right-side-out membrane vesicles prepared from E. coli cells expressing citM. None of the other TCA cycle intermediates and related compounds tested were recognized by CitM. Uptake experiments using radioactive 63Ni2+ provided direct evidence that D-isocitrate is transported in complex with divalent metal ions.
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Cell-wall proteinases PrtS and PrtB have a different role in Streptococcus thermophilus/Lactobacillus bulgaricus mixed cultures in milk
P. Courtin, V. Monnet and F. RulThe manufacture of yoghurt relies on the simultaneous utilization of two starters: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation usually takes place between the two species, which often results in enhanced milk acidification and aroma formation compared to pure cultures. Cell-wall proteinases of Lactococcus lactis and lactobacilli have been shown to be essential to growth in milk in pure cultures. In this study, the role of proteinases PrtS from S. thermophilus and PrtB from Lb. bulgaricus in bacterial growth in milk was evaluated; a negative mutant for the prtS gene of S. thermophilus CNRZ 385 was constructed for this purpose. Pure cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant were made in milk as well as mixed cultures of S. thermophilus and Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant was associated with several strains of Lb. bulgaricus, including a PrtB-negative strain. The pH and growth of bacterial populations of the resulting mixed cultures were followed, and the Lactobacillus strain was found to influence both the extent of the benefit of Lb. bulgaricus/S. thermophilus association on milk acidification and the magnitude of S. thermophilus population dominance at the end of fermentation. In all mixed cultures, the sequential growth of S. thermophilus then of Lb. bulgarius and finally of both bacteria was observed. Although proteinase PrtS was essential to S. thermophilus growth in milk in pure culture, it had no effect on bacterial growth and thus on the final pH of mixed cultures in the presence of PrtB. In contrast, proteinase PrtB was necessary for the growth of S. thermophilus, and its absence resulted in a higher final pH. From these results, a model of growth of both bacteria in mixed cultures in milk is proposed.
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Static growth of mucoid Pseudomonas aeruginosa selects for non-mucoid variants that have acquired flagellum-dependent motility a
More LessaPresent address: Division of Science and Mathematics, University of Minnesota-Morris, Morris, MN 56267, USA.
When mucoid (alginate-producing) Pseudomonas aeruginosa FRD1 is grown under low oxygen conditions in liquid culture (static), non-mucoid variants appear and eventually predominate. This conversion is not readily observed in aerobic, shaken cultures or static cultures containing the alternative electron acceptor nitrate. In this study, it is shown that the non-mucoid variants that arise under static growth conditions are almost exclusively algT mutants. It has been shown that AlgT not only positively regulates alginate biosynthesis, but also directly or indirectly negatively regulates flagellum synthesis. Indeed, during static growth, conversion to the non-mucoid phenotype is accompanied by the acquisition of flagellum-mediated motility. Surprisingly, by using a reporter gene fusion with the fliC promoter (pfliC::xylE), it was found that fliC expression begins within hours of static growth and is reversible after returning the culture to shaking conditions. The ability of the strain to produce alginate seems to be irrelevant to this phenomenon, as an AlgT+ ΔalgD strain showed identical results. Thus, it is suggested that the first effect of static growth is to induce motility as an adaptive measure in the presence of wild-type algT. This may afford P. aeruginosa the ability to swim towards areas of higher oxygen concentrations. Subsequent to this, algT mutations are likely to secure the motile phenotype.
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Novel insights into the interplay between peripheral reactions encoded by xyl genes and the chlorocatechol pathway encoded by tfd genes for the degradation of chlorobenzoates by Ralstonia eutropha JMP134
More LessMany bacteria can grow on chloroaromatic pollutants because they can transform them into chlorocatechols, which are further degraded by enzymes of a specialized ortho-cleavage pathway. Ralstonia eutropha JMP134 is able to grow on 3-chlorobenzoate by using two pJP4-encoded, ortho-cleavage chlorocatechol degradation gene clusters (tfdC I D I E I F I and tfdD II C II E II F II). Very little is known about the acquisition of new catabolic genes encoding enzymes that lead to the formation of chlorocatechols in R. eutropha JMP134. The effect on the catabolic properties of an R. eutropha JMP134 derivative that received the xylS–xylXYZL gene module, encoding the xylS-regulated expression of the broad-substrate-range toluate 1,2-dioxygenase (xylXYZ) and the 1,2-dihydro-1,2-dihydroxytoluate dehydrogenase (xylL) from pWW0, which allows the transformation of 4-chlorobenzoate into 4-chlorocatechol, was studied. Such a derivative could efficiently grow on 4-chlorobenzoate. Unexpectedly, this derivative also grew on 3,5-dichlorobenzoate, a substrate for XylXYZL but not an inducer of the XylS regulatory protein. The ability to grow on 4-chlorobenzoate or 3,5-dichlorobenzoate was also observed in derivatives of strain JMP134 containing the xyl gene module but lacking xylS, indicating the presence of an xylS-like element in R. eutropha with an inducer profile different from that of the pWW0-encoded regulator. Growth on 4-chlorobenzoate was also observed after introduction of the xyl gene module into strain JMP222, a JMP134 derivative lacking pJP4, but only if multiple copies of tfdC I D I E I F I or tfdD II C II E II F II were present. However, only the derivative containing multiple copies of tfdD II C II E II F II was able to grow on 3,5-dichlorobenzoate. These observations indicate that although the acquisition of new catabolic genes actually enhances the catabolic abilities of R. eutropha JMP134, these new properties are strongly influenced by the dosage of the tfd genes, the presence of a chromosomal xylS-like regulatory element and the different contributions of the tfd gene clusters.
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Genome-wide transcriptional profiling of the Bacillus subtilis cold-shock response
More LessThe transcriptome of Bacillus subtilis was analysed at different time points (30, 60 and 90 min) after a temperature downshift from 37 to 18 °C using DNA macroarrays. This approach allowed the identification of around 50 genes exhibiting an increased mRNA level and around 50 genes exhibiting a decreased mRNA level under cold-shock conditions. Many of the repressed genes encode enzymes involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating metabolic adaptation of the cells to the decreased growth rate at the lower temperature. The strongest cold-inducible gene encodes fatty acid desaturase, which forms unsaturated fatty acids from saturated phospholipid precursors, thereby increasing membrane fluidity. The cold-shock-induced increase of mRNA levels of the classical cold-shock genes cspB, cspC and cspD could be verified. Furthermore, besides many genes encoding proteins of unknown function, some genes encoding ribosomal proteins were transcriptionally up-regulated, which points to an adaptive reprogramming of the ribosomes under cold-shock conditions. Interestingly, the amount of mRNA specified by the operon ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB, which encodes enzymes involved in degradation of branched-chain amino acids, also increases after a temperature downshift. As cells utilize the isoleucine and valine degradation intermediates α-methylbutyryl-CoA and isobutyryl-CoA for synthesis of branched-chain fatty acids, this finding reflects the adaptation of membrane lipid composition, ensuring the maintenance of appropriate membrane fluidity at low temperatures. The results of the DNA array analyses were verified for several selected genes by RNA slot-blot analysis and compared with two-dimensional PAGE analyses.
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Transcriptional analysis of the gene encoding peptidyl-tRNA hydrolase in Escherichia coli
More LessGene pth encodes peptidyl-tRNA hydrolase (Pth), an enzyme that cleaves peptidyl-tRNAs released abortively from ribosomes during protein synthesis. In the Escherichia coli chromosome, pth is flanked by ychH and ychF, two genes of unknown function. Pth is essential for cell viability, especially under conditions leading to overproduction of peptidyl-tRNA. In an attempt to unveil the elements that affect pth expression, the transcriptional features of the pth region were investigated. Northern blot experiments showed that both pth and ychF, the 3′-proximal gene, are cotranscribed in a bicistronic transcript. However, transcripts containing each of the individual messages were also detected. Accordingly, two transcriptional promoters were identified by primer extension experiments: one located upstream of pth, which presumably gives rise to both the mono and bicistronic pth transcripts, and the other, preceding ychF, which generates its monocistronic message. Deletion analysis indicates that pth transcript stability depends on ychF integrity. Also, a defect in RNase E activity resulted in Pth overproduction. It is proposed that RNase E processing within ychF in the bicistronic message limits pth expression.
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Catabolism of mannitol in Lactococcus lactis MG1363 and a mutant defective in lactate dehydrogenase
More LessMannitol metabolism in Lactococcus lactis MG1363 and in a derivative strain deficient in lactate dehydrogenase (LDHd) was characterized. Both strains had the ability to grow on mannitol as an energy source, although this polyol was a poorer substrate for growth than glucose. When compared to glucose, the metabolism of mannitol caused an NADH burden due to formation of an additional NADH molecule at the reaction catalysed by mannitol-1-phosphate dehydrogenase (Mtl1PDH). This resulted in a prominent accumulation of mannitol 1-phosphate (Mtl1P) both in growing and resting cells, suggesting the existence of a severe bottleneck at Mtl1PDH. Growth on mannitol induced the activity of Mtl1PDH in both the LDHd and MG1363 strains. The lower accumulation of Mtl1P in mannitol-grown cells when compared to glucose-grown LDHd cells, as monitored by in vivo 13C-NMR, reflects this induction. A clear shift towards the production of ethanol was observed on mannitol, indicating pressure to regenerate NAD+ when this substrate was used. A strategy to obtain a mannitol-overproducing strain is proposed.
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Lack of cable pili expression by cblA-containing Burkholderia cepacia complex a
More LessaThe GenBank accession numbers for the complete cblA nucleotide sequences for the isolates listed in Table 1 are AF455151–AF455162.
The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87·8–88·4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.
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The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae b
bThe GenBank accession number for the waaE gene sequences of P. mirabilis CECT170, Y. enterocolitica R102 and Ent. aerogenes CECT684 reported in this paper are AY075039, AY075041 and AY075040, respectively.
To determine the function of the waaE gene in the biosynthesis of the inner-core LPS of Klebsiella pneumoniae, a waaE non-polar mutant has been constructed. Data obtained from the comparative chemical analysis of LPS samples obtained from the wild-type, the mutant strain and the complemented mutant demonstrated that the waaE gene is involved in substitution of α-L-glycero-D-manno-heptopyranose I (L,D-HeppI) at the O-4 position by a β-D-glucopyranose (β-D-Glcp) residue. In addition, DNA amplification and nucleotide sequence determination studies revealed that waaE homologues located between the waaA and coaD genes are present in clinical isolates of Enterobacteriaceae containing the structure β-D-Glcp-(1→4)-α-L,D-HeppI (K. pneumoniae, Proteus mirabilis and Yersinia enterocolitica), as well as in strains of Serratia marcescens and Enterobacter aerogenes of unknown LPS-core structures. Complementation studies using non-polar waaE mutants prove that all the waaE homologues perform the same function. Furthermore, K. pneumoniae, Ser. marcescens and P. mirabilis non-polar waaE mutants showed reduced adhesion and pathogenicity. In addition, the Ser. marcescens and P. murabilis waaE mutants showed reduced swarming motility and ability to form biofilms in vitro. All these characteristics were rescued by reintroduction of the waaE gene independently of its origin. An easy DNA amplification method to detect this gene was established, which also helps in finding the potential presence of this structural feature [β-D-Glcp-(1→4)-α-L,D-HeppI] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures.
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TonB of Escherichia coli activates FhuA through interaction with the β-barrel a
More LessaThe GenBank accession number for the sequence reported in this paper is Y15319.
FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports Fe3+–ferrichrome and the antibiotics albomycin and rifamycin CGP 4832, and serves as a receptor for the unrelated phages T5, T1, ϕ80 and UC-1, colicin M and microcin J25. The energy source for active transport is the proton-motive force of the cytoplasmic membrane, which is required for all FhuA functions except infection by phage T5, and is thought to be mediated to the outer-membrane receptor FhuA by the TonB protein. The crystal structure of FhuA consists of a β-barrel that is closed by a globular domain. The proximal region carries the TonB box (residues 7–11), for which genetic evidence exists that it interacts with the region around residue 160 of TonB. However, deletion of the TonB box along with the globular domain results in a protein, FhuAΔ5–160, that still displays TonB-dependent active ferrichrome transport across the outer membrane and confers sensitivity to the FhuA ligands. In this study synthetic nonapeptides identical in sequence to amino acids 150–158, 151–159, 152–160, 153–161 and 158–166 of TonB were shown to reduce ferrichrome transport of cells via wild-type FhuA and the corkless derivative FhuAΔ5–160, which suggests that this TonB region is involved in the interaction of TonB with the β-barrel of FhuA. TonB missense mutants reduced the activity of FhuA and FhuAΔ5–160. TonB proteins of different Enterobacteriaceae activated FhuA and FhuAΔ5–160 to a similar degree. TonB of Pantoea agglomerans displayed low activity in an E. coli tonB mutant. Sequencing of the tonB gene of P. agglomerans revealed differences from E. coli TonB in the region around residue 160 of the deduced protein; these differences might contribute to the lower activity of the P. agglomerans TonB protein when coupled to the E. coli FhuA protein. The data support the theory that the β-barrel receives the energy from the cytoplasmic membrane via TonB and responds to the energy input and thus represents the transporting domain of FhuA.
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Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance
More LessThe truB gene of Escherichia coli encodes the pseudouridine-55 (ψ55) synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. A truB null mutant grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37 to 50 °C. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically inactive allele of truB. The temperature sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding an m5U-methyltransferase, modifying the universal U54 tRNA nucleoside, but not by mutations in trmH, encoding the enzyme catalysing the formation of Gm18. The truB mutant proteome contained altered levels of intermediates involved in biogenesis of the outer-membrane proteins OmpA and OmpX. The truB mutation also reduced the basal expression from two σE promoters, degP and rpoHP3. Three novel aspects to the phenotype of truB mutants were identified. Importantly the data support the hypothesis that TruB-effected ψ55 modification of tRNA is not essential, but contributes to thermal stress tolerance in E. coli, possibly by optimizing the stability of the tRNA population at high temperatures.
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The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill b
More LessbThe GenBank accession numbers for the mcrA sequences reported in this paper are AF414034–AF414051 (see Fig. 2) and AF414007–AF414033 (environmental isolates in Fig. 3).
Inferred amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.
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Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima
More LessIn this study, high-affinity maltose- and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity: whereas the maltose-binding activity showed specificity for α-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose- and glucose-binding activity was found in the supernatant retrieved following centrifugation (100000 g ) of the cell-free extracts prepared by French-pressure-cell treatment; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltose-binding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze–thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltose-binding proteins, MalE1 and MalE2, encoded in the genome. The maltose-binding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose- and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.
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Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis b
bThe primer sequences used for PCR in this study are shown as supplementary data on Microbiology Online (http://mic.sgmjournals.org).
GTP-binding proteins are found in all domains of life and are involved in various essential cellular processes. With the recent explosion of available genome sequence data, a widely distributed bacterial subfamily of GTP-binding proteins was discovered, represented by the Escherichia coli Era and the Bacillus subtilis Obg proteins. Although only a limited number of theGTP-binding proteins belonging to the subfamily have been experimentally characterized, and their function remains unknown, the available data suggests that many of them are essential to bacterial growth. When the complete genomic sequence of B. subtilis was surveyed for genes encoding GTP-binding proteins of the Era/Obg family, nine such genes were identified. As a first step in elucidating the functional networks of those nine GTP-binding proteins, data presented here indicates that six of them are essential for B. subtilis viability. Additionally, it is shown that the six essential proteins are able to specifically bind GTP and GDP in vitro. Experimental depletion of the essential GTP-binding proteins was examined in the context of cell morphology and chromosome replication, and it was found that two proteins, Bex and YqeH, appeared to participate in the regulation of initiation of chromosome replication. Collectively, these results suggest that members of the GTP-binding Era/Obg family are important proteins with precise, yet still not fully understood, roles in bacterial growth and viability.
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Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum b
More LessbThe GenBank accession number for the B. japonicum norCBQD genes reported in this paper is AJ132911.
The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (PnorC–lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O2) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The PnorC–lacZ fusion was not expressed in the B. japonicum fixK 2 mutant strain 9043, but complementation of the mutant with the fixK 2 gene restored β-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45·5 bp downstream of the centre of a putative binding site for the transcription factor FixK2.
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NisB is required for the dehydration and NisC for the lanthionine formation in the post-translational modification of nisin
More LessNisin produced by Lactococcus lactis subsp. lactis is a 34-residue antibacterial polypeptide and belongs to a group of post-translationally modified peptides, lantibiotics, with dehydrated residues and cyclic amino acids, lanthionines. These modifications are supposed to be made by enzymes encoded by lanB and lanC genes, found only in biosynthetic operons encoding lantibiotics. To analyse the extent of modification, His-tagged nisin precursors were expressed in nisB and nisC mutant strains. The His-tagged nisin precursors were purified from the cytoplasm of the cells, as lack of NisB or NisC activity impaired translocation of the nisin precursor. The purified His-tagged polypeptides were analysed with trypsin digestion followed by nisin bioassay, SDS-PAGE, N-terminal sequencing and mass spectroscopy. According to the results, nisin precursors from the strain lacking NisB activity were totally unmodified, whereas nisin precursors from the strain lacking NisC activity, but having NisB activity, were dehydrated and devoid of normal lanthionine formation. This is the first experimental evidence showing that NisB is required for dehydration and NisC for correct lanthionine formation in nisin maturation.
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Tn5041-like transposons: molecular diversity, evolutionary relationships and distribution of distinct variants in environmental bacteria b
More LessbThe accession numbers for the nucleotide sequences reported in this work are given in the legends for Figs 1 F1 and 2.
cA comparison of the sequence of INT5041C with other proteins is available as supplementary data on Microbiology Online (http://mic.sgmjournals.org).
A detailed study on the geographic distribution, molecular diversity and evolutionary relationships of 24 closely related variants of the Tn5041 transposon found among 182 mercury resistant environmental Gram-negative strains from the IMG-Hg Reference Collection is reported here. RFLP analysis, followed by the determination of partial DNA sequences, identified 14 distinct types of these transposons, which differed from each other by 1–7 single-event DNA polymorphisms. No polymorphisms were detected at the right arm of the transposons except an insertion of a new mobile DNA element carrying a mer operon (named the mer2 cassette) within the Tn5041 mer operon. According to the model presented here, the insertion occurred via homologous recombination with a circular form of the mer2 cassette. A total of 8 point mutations, 1 internal deletion, 2 end-involving deletions, 3 mosaic regions and 2 insertions were detected at the left arm of the transposons. The insertions were a transposon closely related to Tn21 but lacking the integron and a new group II intron (named INT5041C). Inspection of the geographic distribution of the Tn5041 variants suggested that at least three long-distance waves of dissemination of these variants had occurred, accompanied by homologous recombination between different Tn5041 lineages. Movements of circular DNAs by homologous recombination as a source of mosaic genes and new mer genes, and formation of unusual mosaics ending or beginning at the Tn5041 att site are discussed.
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A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa
Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA–recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN6TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN6TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA–recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.
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cis-Acting elements that regulate the low-pH-inducible urease operon of Streptococcus salivarius
More LessDifferential expression of the Streptococcus salivarius 57.I urease operon in response to pH is effected by repression of transcription from a proximal promoter, PureI. To localize the cis-acting elements involved in the regulation of the urease operon, the intact promoter region and its derivatives were generated and fused to a promoterless chloramphenicol acetyltransferase (cat) gene. The promoter–cat fusions were established in the lacZ gene of S. salivarius by using a newly constructed integration vector. CAT-specific activities were examined in batch-grown cells at pH 7·5 and 5·5. The results indicated that a 21 bp region immediately 5′ to the −35 element was required for efficient repression of PureI at neutral pH and that the 39 bp (−57 to −95) 5′ to this region contained sequences required for optimal expression of PureI. A potential secondary repressor-binding site was tentatively identified further upstream of the −35 element (−96 to −115). To further analyse the cis-acting elements, base changes were introduced into two AT-rich repeats within the primary repressor-binding site. One such derivative, S. salivarius M1, with five base substitutions immediately 5′ to the −35 element, expressed 20-fold more CAT-specific activity at neutral pH than the strain carrying wild-type PureI–cat. Also, the pH sensitivity of strain M1 was greatly reduced, suggesting that this AT-rich region is crucial for repression of the urease operon. Deletion of three consecutive 15- or 16-base segments from −52 to −96 in the S. salivarius M1 background resulted in lower activities compared to strain M1, confirming the presence of sequences required for optimal expression of the operon. All of the PureI–cat fusions were also integrated into the gtfG gene of Streptococcus gordonii DL1, a non-ureolytic oral Streptococcus sp. Repression of PureI was observed at neutral pH in S. gordonii and the effects of the various mutations of the repressor-binding site largely paralleled those seen in S. salivarius, suggesting that the cis-elements may be a target for a global regulatory circuit that controls gene expression in streptococci in response to pH.
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The role of multiple SOS boxes upstream of the Mycobacterium tuberculosis lexA gene – identification of a novel DNA-damage-inducible gene
More LessFour potential binding sites for LexA were identified upstream of the Mycobacterium tuberculosis lexA gene. A mutational analysis of these sites in a lexA–lacZ reporter construct revealed that only one of these SOS boxes was required for DNA-damage-mediated regulation of lexA expression. A novel DNA-damage-inducible gene, Rv2719c, was identified that was divergently transcribed relative to lexA; the other three SOS boxes were found to be involved in regulating expression of this novel mycobacterial-specific gene. The SOS boxes lay in the respective promoter regions of the genes that they regulated.
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Molecular analysis of the soluble butane monooxygenase from ‘Pseudomonas butanovora’ a
More LessaThe GenBank accession number for the bmoXYBZDC sequence is AY093933.
‘Pseudomonas butanovora’ is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation. The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster. Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB. A sixth structural gene (bmoD) encodes a 9·6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases. Insertional inactivation of bmoX resulted in a mutant ‘P. butanovora’ strain incapable of growth with butane. A putative promoter element characteristic of promoters associated with σ54-dependent transcription initiation was located upstream of the bmo genes. Expression of all six genes was detected in butane-induced cells. Butane monooxygenase from ‘P. butanovora’ aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif. The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.
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Specificity of respiratory pathways involved in the reduction of sulfur compounds by Salmonella enterica
More LessThe tetrathionate (Ttr) and thiosulfate (Phs) reductases of Salmonella enterica LT2, together with the polysulfide reductase (Psr) of Wolinella succinogenes, are unusual examples of enzymes containing a molybdopterin active-site cofactor since all formally catalyse sulfur–sulfur bond cleavage. This is in contrast to the oxygen or hydrogen transfer reactions exhibited by other molybdopterin enzymes. Here the catalytic specificity of Ttr and Phs has been compared using both physiological and synthetic electron-donor systems. Ttr is shown to catalyse reduction of trithionate but not sulfur or thiosulfate. In contrast, Phs cannot reduce tetrathionate or trithionate but allows whole cells to utilize elemental sulfur as an electron acceptor. Mechanisms are proposed by which the bacterium is able to utilize an insoluble sulfur substrate by means of reactions at the cytoplasmic rather than the outer membrane.
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Legionella pneumophila induces apoptosis via the mitochondrial death pathway
More LessLegionella pneumophila has been shown to induce apoptosis within macrophages, monocytic cell lines and alveolar epithelial cells. The mechanisms and significance of L. pneumophila-associated apoptosis are not well understood. It has been speculated that L. pneumophila may induce apoptosis through ligation of death receptors by bacterial surface components or by secreted bacterial factors. Translocation of apoptotic factor(s) through the Dot/Icm secretion machinery followed by direct activation of caspases within the cytosol is discussed as another possible mechanism of apoptosis induction by L. pneumophila. Here, it is shown that L. pneumophila induced the mitochondrial release of cytochrome c in CD95 (Fas/Apo-1)-negative monocytic Mono Mac 6 cells, indicating that Legionella-induced apoptosis is mediated via the mitochondrial signalling pathway. In addition, blocking of the death receptor pathway at distinct stages using CD95-, FADD- or caspase-8-deficient Jurkat cells did not affect induction of apoptosis by L. pneumophila. Conversely, inhibition of the mitochondrial death pathway by overexpression of the anti-apoptotic protein Bcl-2 potently inhibited the processing of caspases and the induction of apoptosis. Therefore, these findings support a model in which the induction of apoptosis by L. pneumophila is mediated by activation of the intrinsic mitochondrial death pathway in the absence of external death receptor signalling.
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Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria b
bThe GenBank accession numbers for the clone sequences reported in this paper can be found in Table 1 T1 ; the accession number for isolate MIB-CB3 is AJ418059.
Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga–Flavobacter–Bacteroides phylum, which the authors named ‘mouse intestinal bacteria’. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale–Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).
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Comparative studies of immunity proteins of pediocin-like bacteriocins
More LessGenes encoding pediocin-like bacteriocins are usually co-transcribed with a gene encoding a cognate immunity protein. To investigate the functionality and specificity of immunity proteins, immunity genes belonging to the bacteriocins curvacin A, enterocin A, enterocin P, leucocin A, pediocin PA-1 and sakacin P, as well as a putative immunity gene, orfY, were expressed in three bacteriocin-sensitive lactic acid bacteria (Lactobacillus sake, Carnobacterium piscicola and Enterococcus faecalis). The transformed indicator strains, each containing one of the immunity genes, were tested for sensitivity towards seven different purified bacteriocins (curvacin A, enterocin A, enterocin P, leucocin A, leucocin C, pediocin PA-1 and sakacin P). Cross-immunity was observed almost exclusively in situations where either the bacteriocins or the immunity proteins belonged to the same sequence-based subgroup. In a few cases, the functionality of immunity proteins was strain-dependent; e.g. the leucocin A immunity gene provided immunity to enterocin A, pediocin PA-1 and leucocin A in Ent. faecalis, whereas in the other two indicators, this gene provided immunity to leucocin A only. The orfY gene, which is transcribed without a cognate bacteriocin, was shown to encode a functional immunity protein that expands the bacteriocin resistance of the strain possessing this gene. The results show that the bacteriocin sensitivity of a lactic acid bacterium strain can depend on (1) the presence of immunity genes in connection with its own bacteriocin production, (2) the presence of extra immunity genes and (3) more general properties of the strain such as the membrane composition or the presence of receptors.
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Identification of strain-specific genes located outside the plasticity zone in nine clinical isolates of Helicobacter pylori b
bThe GenBank accession numbers for the H. pylori sequences reported in this paper are AF326599–AF326607 for region A, AF326608–AF326616 for region B, AF326617–AF326625 for region C, AF326626–AF326634 for region D, AF327212–AF327220 for region E, AF328909–AF328916 and AF328924 for region F, and AF32917–AF32923 for region G.
Helicobacter pylori is a Gram-negative bacterium that is associated with the development of peptic ulcers and gastric carcinoma in humans. This species appears to be one of the most genetically variable bacteria described to date. The overall level of heterogeneity within strains of this organism was determined by comparing the genome sequences of two reference strains, J99 and 26695. The aim of this study was to measure the genetic diversity within strains of H. pylori by looking for strain-specific genes in nine H. pylori strains isolated from patients suffering from chronic gastritis (n=3), duodenal ulcers (n=3) or gastric cancer (n=3). Seven loci that contained strain-specific genes in strains J99 and 26695 were studied. These regions were subsequently amplified from most of the clinical isolates studied and their sequences were determined. ORFs were predicted from the sequence data and were compared to sequences within the databases. The results showed that the genes flanking the ORFs specific to either strain J99 or strain 26695 were also present in a similar configuration in the genomes of the nine clinical isolates. Moreover, in most regions, ORFs homologous to those found in the corresponding loci in the two reference strains were detected. However, in 10 regions, genes similar to those located at another locus in the genome of J99 or 26695 were found. Finally, six strain-specific genes were identified in three regions of three of the H. pylori strains isolated from patients with duodenal ulcers (n=2) and gastric cancer (n=1). Of these six genes, five were putative genes and one was an orthologue of a gene encoding a transposase in Thermotoga maritima. However, no association with disease was found for these genes.
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Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates
More LessAcquisition of virulence genes encoded on mobile genetic elements has played an important role in the emergence of pathogenic isolates of Vibrio cholerae, the causative agent of the diarrhoeal disease cholera. The genes encoding cholera toxin (ctxAB), the main cause of profuse secretory diarrhoea in cholera, are encoded on a filamentous bacteriophage CTXϕ. The toxin coregulated pilus (TCP), an essential intestinal colonization factor, was originally designated as part of a pathogenicity island named the Vibrio pathogenicity island (VPI), but this island has more recently been proposed to be the genome of a filamentous phage, VPIϕ. In this study, it is shown that nanH, which encodes neuraminidase, maps within a novel pathogenicity island designated VPI-2. The 57·3 kb VPI-2 has all of the characteristic features of a pathogenicity island, including the presence of a bacteriophage-like integrase (int), insertion in a tRNA gene (serine) and the presence of direct repeats at the chromosomal integration sites. Additionally, the G+C content of VPI-2 (42 mol%) is considerably lower than that of the entire genome (47 mol%). VPI-2 encodes several gene clusters, such as a restriction modification system (hsdR and hsdM) and genes required for the utilization of amino sugars (nan-nag region) as well as neuraminidase. To determine the distribution of VPI-2 among V. cholerae, 78 natural isolates were examined using PCR and Southern hybridization analysis for the presence of this region. All toxigenic V. cholerae O1 serogroup isolates examined contained VPI-2, whereas non-toxigenic isolates lacked the island. Of 14 V. cholerae O139 serogroup isolates examined, only one strain, MO2, contained the entire 57·3 kb island, whereas 13 O139 isolates contained only a 20·0 kb region with most of the 5′ region of VPI-2 which included nanH deleted in these strains.
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Peptide methionine sulfoxide reductase (MsrA) is not a major virulence determinant for the oral pathogen Actinobacillus actinomycetemcomitans a
More LessaThe GenBank accession number for the msrA sequence reported in this paper is AY026361.
Actinobacillus actinomycetemcomitans is an oral pathogen that is a causative agent for periodontal disease as well as other non-oral infections. The chronic inflammation associated with periodontal diseases suggests that the bacterium must be able to neutralize oxygen intermediates to survive in the host tissues. Methionine sulfoxide reductase (MsrA) is an enzyme that has been demonstrated to have a role in protection against oxidative damage and has also been identified to be required for the proper expression or maintenance of functional adhesins on the surface of several pathogenic bacteria. The A. actinomycetemcomitans homologue of msrA has been isolated and a chromosomal insertion mutant constructed by allele replacement mutagenesis. Inactivation of the gene led to a complete loss of enzymic activity toward a synthetic substrate. However, the isogenic mutant was not more sensitive to oxidative stress or less adherent to epithelial cells as compared with the parent strain. These data suggest that this strain of A. actinomycetemcomitans has redundant systems that compensate for the MsrA activities ascribed for other organisms.
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Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence
More LessCopper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is suspected to be one of the anti-oxidant enzymes and virulence determinants active in some pathogenic micro-organisms. To elucidate the role of Cu/ZnSOD in the major human fungal pathogen Candida albicans, its gene, designated SOD1, was disrupted by the URA-blaster technique. The resulting sod1/sod1 mutant showed delayed hyphal growth on Spider medium but could still form hyphae on other solid media or in liquid media, particularly in response to serum. Moreover, the sod1/sod1 mutant was more sensitive to menadione, a redox-cycling agent, than the isogenic wild-type strain, although it still showed an adaptive oxidative stress response. Furthermore, the sod1/sod1 mutant cells exhibited slow growth in minimal medium when compared to the wild-type cells, but their growth was restored by the addition of lysine to the medium. Interestingly, C. albicans cells lacking Cu/ZnSOD showed increased susceptibility to macrophage attack and had attenuated virulence in mice. Thus, these results suggest that Cu/ZnSOD is required for the protection of C. albicans against oxidative stresses and for the full virulence of the organism to be expressed.
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Cycles of mitochondrial energization driven by the ultradian clock in a continuous culture of Saccharomyces cerevisiae
More LessA continuous culture of Saccharomyces cerevisiae IFO 0233, growing with glucose as the major carbon and energy source, shows oscillations of respiration with a period of 48 min. Samples taken at maxima and minima indicate that (i) periodic changes do not occur as a result of carbon depletion, (ii) intrinsic differences in respiratory activity occur in washed organisms and (iii) a respiratory inhibitor accumulates during respiratory oscillations. Plasma membrane and inner mitochondrial membranes generate transmembrane electrochemical potentials; changes in these can be respectively assessed using anionic or cationic fluorophores. Thus flow cytometric analyses indicated that an oxonol dye [DiBAC4(3); bis(1,3-dibutylbarbituric acid)trimethine oxonol] was excluded from yeasts to a similar extent (in >98% of the population) at all stages, showing that the plasma membrane potential was maintained at a steady value. However, uptake of Rhodamine 123 was greatest at that phase characterized by a low respiratory rate. Addition of uncouplers of energy conservation [CCCP (m-chlorocarbonylcyanide phenylhydrazone) or S-13(5-chloro-3-t-butyl-2-chloro-41-nitrosalicylanilide)] to the continuous cultures increased the respiration, but had only a transient effect on the period of the oscillation. Electron microscopy showed changes in mitochondrial ultrastructure during the respiratory oscillation. At low respiration the cristae were more clearly defined due to swelling of the matrix; this corresponds to the ‘orthodox’ conformation. When respiration was high the mitochondrial configuration was ‘condensed’. It has been shown previously that a temperature-compensated ultradian clock operates in S. cerevisiae. It is proposed that mitochondria undergo cycles of energization in response to energetic demands driven by this ultradian clock output.
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YlBMH1 encodes a 14-3-3 protein that promotes filamentous growth in the dimorphic yeast Yarrowia lipolytica a
More LessaThe GenBank accession numbers for the YlBMH1 and YlBMH2 sequences reported in this study are AY090661 and AY090662, respectively.
Most pathogenic fungi have the ability to alternate between a unicellular yeast form and different filamentous forms (hyphae and pseudohyphae). This attribute is generally regarded as an important virulence factor and has also attracted attention because of its implications in the study of eukaryotic cell differentiation. To identify genes that are involved in the regulation of these events, chemical mutagenesis of the dimorphic yeast Yarrowia lipolytica was performed and morphological mutants that were unable to form hyphal cells were isolated. Screening of a Y. lipolytica genomic DNA library for genes able to complement this defect led to the isolation of YlBMH1, a gene encoding a 14-3-3 protein and whose transcription levels are increased during the yeast-to-hypha transition. Remarkably, overexpression of YlBMH1 was able to enhance pseudohyphae formation in a strain lacking functional YlRAC1 but caused no visible effects in Δmhy1 and Δbem1 cells, thus suggesting that YlBMH1 is involved in the regulation of both hyphal and pseudohyphal growth in Y. lipolytica. The identification of YlBMH2, a gene encoding a second 14-3-3 protein (YlBmh2p) that contains a 19 aa insertion absent in all other members of the 14-3-3 family, is also reported. Differently from YlBMH1, the transcription levels of YlBMH2 do not show any apparent variation during the induction of hyphal growth, and its overexpression has no effects on cells lacking functional MHY1, YlRAC1 or YlBEM1. Taken together, these observations suggest that, in spite of their high conservation, YlBmh1p and YlBmh2p have different cellular functions.
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Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus
Muscodor vitigenus is a recently described endophytic fungus of Paullinia paullinioides, a liana growing in the understorey of the rainforests of the Peruvian Amazon. This fungus produces naphthalene under certain cultural conditions. Naphthalene produced by M. vitigenus was identified by gas chromatography/mass spectrometry. Its chromatographic and mass spectral properties were identical to authentic naphthalene. Agar plugs supporting growth of the fungus and producing known amounts of naphthalene effectively repelled the adult stage of the wheat stem sawfly, Cephus cinctus, in Y-tube bioassay tests. Authentic naphthalene, at comparable concentrations to those in tests involving the fungus itself, mimicked the insect repellency of the fungus. Although other Muscodor spp. produce volatile antimicrobials, M. vitigenus is unique in its ability to produce naphthalene almost exclusively. This report also describes the potential practical implications of M. vitigenus.
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Differential secretion of Sap4–6 proteins in Candida albicans during hyphae formation
More LessSecreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multi-gene family and are considered to be putative virulence factors for candidiasis. SAP4–6 mRNAs were first detected during hyphae formation and were assumed to play roles in the development of disseminated candidiasis. Recombinant Sap proteins (Sap2–6) were prepared and specific antibodies were generated against Sap2–6. The presence of Sap4, Sap5 and Sap6, but not Sap2 or Sap3, was demonstrated in culture supernatants of C. albicans after induction of hyphae formation. In parallel to hyphae formation, Sap5 (∼40 kDa) was detected as early as ∼6 h after induction at neutral pH, and Sap4/6 (∼43 kDa) were detected after ∼24 h when the culture medium became acidic. The differential secretion of Sap5 and Sap4/6 was affected when the culture medium pH was buffered at pH 6·5 or pH 4·5. In addition, intracellular pools of Sap4–6 seem to exist, and protein is not necessary for Sap4–6 induction. This study provides the first evidence that Sap4–6 proteins in C. albicans are differentially produced and secreted during hyphae formation.
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)