- Volume 147, Issue 6, 2001
Volume 147, Issue 6, 2001
- Genetics And Molecular Biology
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Expression of Streptococcus mutans fimA is iron-responsive and regulated by a DtxR homologue
More LessIron uptake, transport and storage in Streptococcus mutans, the principal causative agent of human dental cavities, is unexplored despite early reports in the literature which predict a role for this trace metal in cariogenesis. Experiments in the authors’ laboratory revealed several iron-responsive proteins in S. mutans, one of which reacted with a polyclonal antiserum directed against the FimA fimbrial adhesin from Streptococcus parasanguis on Western blots. The results of Western blot and Northern hybridization experiments support an inverse relationship between iron availability and S. mutans fimA expression, and metal ion uptake experiments implicate FimA in S. mutans 55Fe transport. Cloning of the S. mutans fimA homologue facilitated the construction of a fimA knockout mutant which grew poorly in an iron-limiting medium relative to the wild-type progenitor strain, lending further support to a role for FimA in S. mutans iron transport. The authors also identified and cloned a dtxR-like gene (dlg) located downstream of fimA on the S. mutans chromosome, and noted increased fimA expression in a S. mutans dlg knockout mutant relative to wild-type on RNA spot blots and Western blots. The uptake of 55Fe, which was also significantly increased in this mutant, was compromised in a fimA/dlg double knockout. These findings are consistent with a role for Dlg in the iron-mediated regulation of fimA, and possibly other S. mutans iron transporters. Finally, the cariogenic potential of the fimA and dlg knockout mutants was not significantly different from that of the wild-type progenitor in a germ-free rat model.
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Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358
More LessThe GenBank/EMBL/DDBJ accession numbers for the pcaR, pobC-pobA and pcaHG sequences reported in this paper are AJ252090, AJ251792 and AJ295623, respectively.
The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the β-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB. In this study the identification and characterization of the pobC-pobA locus of P. putida WCS358 is presented. The p-hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated PobC. Using the pobA promoter transcriptionally fused to a promoterless lacZ gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA. This PobC-PCA response was genetically demonstrated by making use of pobC::Tn5 and pcaH::Tn5 mutants of strain WCS358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed. Finally, the activation of pobA by PHB varied according to the concentration and it was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the β-ketoadipate pathway, one of which is pcaK. It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.
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Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes
The EMBL accession numbers for the sequences reported in this paper are AJ245436 [P. putida (oleovorans) GPo1 alk gene clusters and flanking DNA], AJ233397 (P. putida P1 alk gene clusters and flanking DNA), AJ249793 (P. putida P1 nahKJ genes), AJ249825 [P. putida (oleovorans) GPo1 16S RNA gene] and AJ271219 (P. putida P1 16S RNA gene).
The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9·7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80–92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind AlkS.
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- Genomics
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Sulfur-limitation-regulated proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study
Little is known about the genes and enzymes involved in sulfur assimilation in Bacillus subtilis, or about the regulation of their expression or activity. To identify genes regulated by sulfur limitation, the authors used two- dimensional (2D) gel electrophoresis to compare the proteome of a wild-type strain grown with either sulfate or glutathione as sole sulfur source. A total of 15 proteins whose synthesis is modified under these two conditions were identified by matrix-assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry. In the presence of sulfate, an increased amount of proteins involved in the metabolism of C1 units (SerA, GlyA, FolD) and in the biosynthesis of purines (PurQ, Xpt) and pyrimidines (Upp, PyrAA, PyrF) was observed. In the presence of glutathione, the synthesis of two uptake systems (DppE, SsuA), an oxygenase (SsuD), cysteine synthase (CysK) and two proteins of unknown function (YtmI, YurL) was increased. The changes in expression of the corresponding genes, in the presence of sulfate and glutathione, were monitored using slot-blot analyses and lacZ fusions. The ytmI gene is part of a locus of 12 genes which are co-regulated in response to sulfur availability. This putative operon is activated by a LysR-like regulator, YtlI. This is the first regulator involved in the control of expression in response to sulfur availability to be identified in B. subtilis.
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- Pathogenicity And Medical Microbiology
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The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems
More LessThe GenBank accession numbers for the DNA sequences of the rpoS loci in E. coli strains HU734 and CFT073 are AF275947 and AF270497, respectively.
Trehalose synthesis (RpoS-dependent) and betaine uptake mediated by transporters ProP and ProU contribute to the osmotolerance of Escherichia coli K-12. Pyelonephritis isolates CFT073 and HU734 were similar and diminished in osmotolerance, respectively, compared to E. coli K-12. The roles of RpoS, ProP and ProU in osmoregulation and urovirulence were assessed for these isolates. Strain HU734 expressed an RpoS variant which had low activity and a C-terminal extension. This bacterium accumulated very little trehalose and had poor stationary-phase thermotolerance. For E. coli CFT073, introduction of an rpoS deletion impaired trehalose accumulation, osmotolerance and stationary-phase thermotolerance. The rpoS defects accounted for the difference in osmotolerance between these strains in minimal medium of very high osmolality (1·4 mol kg−1) but not in medium of lower osmolality (0·4 mol kg−1). The slow growth of both pyelonephritis isolates in high-osmolality medium was stimulated by glycine betaine (GB) and deletion of proP and/or proU impaired GB uptake. An HU734 derivative lacking both proP and proU retained osmoprotective GB uptake activity that could be attributed to system BetU, which is not present in strain K-12 or CFT073. BetU transported GB (K m, 22 μM) and proline betaine. High-osmolality human urine (0·92 mol kg−1) included membrane-permeant osmolyte urea (0·44 M) plus other constituents which contributed an osmolality of only approximately 0·4 mol kg−1. Strains HU734 and CFT073 showed correspondingly low GB uptake activities after cultivation in this urine. Deletion of proP and proU slowed the growth of E. coli HU734 in this high-osmolality human urine (which contains betaines) but had little impact on its colonization of the murine urinary tract after transurethral inoculation. By contrast, deletion of rpoS, proP and proU had no effect on the very rapid growth of CFT073 in high-osmolality urine or on its experimental colonization of the murine urinary tract. RpoS-dependent gene expression is not essential for growth in human urine or colonization of the murine urinary tract. Additional osmoregulatory systems, some not present in E. coli K-12 (e.g. BetU), may facilitate growth of pyelonephritis isolates in human urine and colonization of mammalian urinary tracts. The contributions of systems ProP and ProU to urinary tract colonization cannot be definitively assessed until all such systems are identified.
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Commensal Escherichia coli isolates are phylogenetically distributed among geographically distinct human populations
An intraspecies phylogenetic grouping of 168 human commensal Escherichia coli strains isolated from the stools of three geographically distinct human populations (France, Croatia, Mali) was generated by triplex PCR. The distributions of seven known extraintestinal virulence determinants (ibeA, pap, sfa/foc, afa, hly, cnf1, aer) were also determined by PCR. The data from the three populations were compiled, which showed that strains from phylogenetic groups A (40%) and B1 (34%) were the most common, followed by phylogenetic group D strains (15%). Strains of the phylogenetic group B2 were rare (11%). However, a significant specific distribution for strains of groups A, B1 and B2 within each population was observed, which may indicate the influence of (i) geographic/climatic conditions, (ii) dietary factors and/or the use of antibiotics or (iii) host genetic factors on the commensal flora. Virulence determinants were rarely detected, with only 25·6% of the strains harbouring at least one of the virulence genes tested. The strains with virulence factors most frequently belonged to phylogenetic group B2. The commensal strains of phylogenetic groups A, B1 and D had fewer virulence determinants than pathogenic strains of the corresponding groups when these data were compared with those for previous collections of virulent extraintestinal infection strains studied using the same approach. However, the virulence patterns of commensal and pathogenic B2 phylogenetic group strains were the same. The data thus suggest that strains of the A, B1 and D phylogenetic groups predominate in the gut flora and that these strains must acquire virulence factors to become pathogenic. In contrast, commensal phylogenetic group B2 strains are rare but appear to be potentially virulent.
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- Physiology And Growth
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Metabolic flux in cellulose batch and cellulose-fed continuous cultures of Clostridium cellulolyticum in response to acidic environment
More LessClostridium cellulolyticum, a nonruminal cellulolytic mesophilic bacterium, was grown in batch and continuous cultures on cellulose using a chemically defined medium. In batch culture with unregulated pH, less cellulose degradation and higher accumulation of soluble glucides were obtained compared to a culture with the pH controlled at 7·2. The gain in cellulose degradation achieved with pH control was offset by catabolite production rather than soluble sugar accumulation. The pH-controlled condition improved biomass, ethanol and acetate production, whereas maximum lactate and extracellular pyruvate concentrations were lower than in the non-pH-controlled condition. In a cellulose-fed chemostat at constant dilution rate and pH values ranging from 7·4 to 6·2, maximum cell density was obtained at pH 7·0. Environmental acidification chiefly influenced biomass formation, since at pH 6·4 the dry weight of cells was more than fourfold lower compared to that at pH 7·0, whereas the specific rate of cellulose assimilation decreased only from 11·74 to 10·13 milliequivalents of carbon (g cells)−1 h−1. The molar growth yield and the energetic growth yield did not decline as pH was lowered, and an abrupt transition to washout was observed. Decreasing the pH induced a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation. The acetate/ethanol ratio decreased as the pH declined, reaching close to 1 at pH 6·4. Whatever the pH conditions, lactate dehydrogenase was always greatly in excess. As pH decreased, both the biosynthesis and the catabolic efficiency of the pyruvate-ferredoxin oxidoreductase declined, as indicated by the ratio of the specific enzyme activity to the specific metabolic rate, which fell from 9·8 to 1·8. Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 6·2. C. cellulolyticum appeared to be similar to rumen cellulolytic bacteria in its sensitivity to acidic conditions. Apparently, the cellulolytic anaerobes studied thus far do not thrive when the pH drops below 6·0, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes.
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An NMR and enzyme study of the carbon metabolism of Neisseria meningitidis
More LessThe pathogenic neisseriae are fastidious bacteria that are only able to grow on a restricted range of carbon sources. The genome sequence of Neisseria meningitidis strain MC58 predicts the presence of a complete citric acid cycle (CAC), but there have been no detailed biochemical studies of carbon metabolism in this important pathogen. In this study, both NMR and conventional enzyme assays were used to investigate the central metabolic pathways of a serogroup B strain (K454). 13C-NMR labelling patterns of amino acids from hydrolysed cell proteins after growth with either 2- or 3-[13C]pyruvate were consistent with the operation of a complete oxidative CAC. Enzyme assays showed that cell-free extracts contained all the CAC enzymes predicted from the genome sequence, including a membrane-bound malate:quinone oxidoreductase which is present in place of the conventional NAD-linked cytoplasmic malate dehydrogenase. 1H-NMR studies showed that growth on glucose, lactate and, especially, pyruvate, resulted in the excretion of significant amounts of acetate into the culture supernatant. This occurred via the phosphotransacetylase (PTA)–acetate kinase (ACK) pathway. Extremely high specific activities of PTA (7–14 μmol min−1 mg−1) were detected in cell-free extracts, although ACK activities were much lower (46–298 nmol min−1 mg−1). Expression of PTA and ACK activities was not co-ordinately regulated during growth on combinations of carbon sources. This may be related to the presence of two ackA paralogues in N. meningitidis which are, unusually, unlinked to the pta gene.
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Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli
More LessThe metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO2, was assessed using an isogenic set of genetically engineered strains of Escherichia coli. In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the poxB gene was expressed constitutively or from the IPTG-inducible tac promoter. Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (0·05 h−1) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 0·25 h−1) unless expressed constitutively or from the tac promoter. The poor complementation of PDH-deficient strains by poxB plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural poxB promoter and deleterious effects of the multicopy plasmids. As a consequence of replacing the PDH complex by PoxB, the growth rate (μmax), growth yield (Y max) and the carbon conversion efficiency (flux to biomass) were lowered by 33%, 9–25% and 29–39% (respectively), indicating that more carbon has to be oxidized to CO2 for energy generation. Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose. In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of E. coli. In glucose minimal medium, the respective growth rates (μmax), growth yields (Y max) and carbon conversion efficiencies were 16%, 14% and 24% lower than the parental values, and correspondingly more carbon was fluxed to CO2 for energy generation. It was concluded that PoxB is used preferentially at low growth rates and that E. coli benefits from being able to convert pyruvate to acetyl-CoA by a seemingly wasteful route via acetate.
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- Systematics And Evolution
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A novel filamentous Bacillus sp., strain NAF001, forming endospores and budding cells
The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence reported in this paper is AB049195.
A novel filamentous bacterium, strain NAF001, was isolated from suspended water of a domestic wastewater treatment tank. It formed an extremely long filamentous trichome and produced endospores. It formed spore-like resting cells (SLRCs) which were heat-resistant. SLRCs grew by budding to form short filaments resembling the gonidia of filamentous bacteria such as Leucothrix. This is the first report of a Bacillus species that exhibits budding growth. The filamentous form was neither restricted to any particular growth stage nor dependent on cultural conditions. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Bacillus, with no close relatives at the species level (sequence similarity <95·3%). Strain NAF001 thus probably belongs to a new and novel species of Bacillus.
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Evidence for a more recently evolved clade within a Candida albicans North American population
More LessCandida albicans is diploid and displays a primarily clonal mode of reproduction. There is, however, evidence for meiosis and the degree to which this occurs in nature is unknown. Although random mating would act to obscure clonal lineages, previous studies have demonstrated that collections of North American isolates display three major partitions with no evidence of geographic clustering. To better understand the extent of sexuality and its role in the phylogeny of the species, a reference subset of 50 isolates representing this tripartite division was analysed using 1 minisatellite, 5 microsatellites (MSs) and 15 nuclear polymorphisms (NP). A total of 87 alleles were observed for 21 loci and 12/16 informative loci exhibited a departure from Hardy–Weinberg expectations (G 2≤0·05). We did not observe an absolute correlation between MSs and NP, although isolates with identical NP genotypes were correlated with a previously defined, predominant class (putative group I). The use of additional markers did not give increased support for the tripartite structure of the population. However, (9/19) group I isolates were found to be highly related, differing by only one or a few alleles. Designated subgroup A, the interpretation is that these isolates are related by descent and that they are of a more recent evolutionary origin, diverging from an ancestral group I clone. The reason for their relative abundance in the population is unknown; one possibility is that they may be under positive selection.
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Population genetics of Helicobacter pylori in the southern part of Switzerland analysed by sequencing of four housekeeping genes (atpD, glnA, scoB and recA), and by vacA, cagA, iceA and IS605 genotyping
The GenBank accession numbers for the sequences reported in this paper are AY004351–AY004662
The population biology of 78 Helicobacter pylori strains (71 from Swiss Italian, 4 from East Asian and 3 from South African patients) was investigated by sequence analysis of four housekeeping genes: atpD, scoB, glnA and recA. The vacA genotype, the presence of cagA and IS605, the iceA allelic type, and the resistance to metronidazole, clarithromycin and amoxycillin were determined. A high percentage of DNA polymorphic sites (19·8% for atpD, 21·3% for scoB, 23·7% for glnA and 20·3% for recA) was found. The phylogenetic trees based on the nucleotide sequences of the four gene fragments showed different topologies and were incongruent. The virulence-associated markers were distributed over the dendrograms and no association was found with phylogenetic clusters or clinical manifestations (chronic gastritis, gastric or duodenal ulcer, MALT lymphoma). Moreover, the H ratios (calculated with the homoplasy test) ranged from 0·742 to 0·799, depending on the gene fragment examined. All these observations suggest that H. pylori exists as a recombinant population. The clustering of the strains according to their geographical origin (USA/Europe, East Asia, South Africa) that has recently been demonstrated elsewhere could only be confirmed for the East Asian vacA s1c strains. In contrast, the South African strains clustered together only in the atpD tree. Presumably, recombination at the different loci has masked the evolutionary relationship among the strains.
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