- Volume 151, Issue 3, 2005
Volume 151, Issue 3, 2005
- Review
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The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together
More LessThe cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as ‘classical’ cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of ‘non-classical’ secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based ‘supramolecular construction kits' in nanobiotechnology.
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- Microbiology Comment
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- Cell And Developmental Biology
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The interplay of glycogen metabolism and differentiation provides an insight into the developmental biology of Streptomyces coelicolor
More LessMycelial colonies of the developmentally complex actinomycete Streptomyces coelicolor growing on solid medium contain glycogen in two distinct locations. Phase I deposits are found in a substrate mycelium region bordering the developing aerial mycelium. Their production involves GlgBI, one of two glycogen branching enzyme isoforms. Phase II deposits occur in the upper regions of aerial hyphae, in long tip cells that are dividing, or have just divided, into unigenomic prespore compartments. Their formation involves a second branching enzyme isoform, GlgBII. To find out if the gene for the second isoform, glgBII, is regulated by any of the well-studied whiA, B, G, H or I genes needed for sporulation septation, glgBI or glgBII was disrupted in a set of whi mutants, and the glycogen phenotypes examined by transmission electron microscopy. In the whiG mutants, deposits were found throughout the aerial mycelium and the adjacent region of the substrate mycelium, but the morphology of all the deposits, i.e. whether they were in the form of granules of branched glycogen or large blobs of unbranched glycan, depended solely on GlgBI. In contrast, the whiA, B, H and I mutations had no obvious effect on the pattern of glycogen deposition, or on the spatial specificity of the branching enzyme isoforms (though phase II glycogen deposits were reduced in size and abundance in the whiA and B mutants, and increased in the whiH mutant). These results indicate that glgBII is regulated (directly or indirectly) by whiG, and not by any of the other whi genes tested, and that the aerial hyphae of a whiG mutant are atypical in being physiologically similar to the substrate hyphae from which they emerge. A new role for aerial hyphae is proposed.
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Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis
More LessDuring Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.
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- Biochemistry And Molecular Biology
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Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA
More LessParacoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)−1. The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3·3 mkat (kg protein)−1]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (α) and 42 kDa (β) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores.
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Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus
More LessGram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might be significantly different from that in Gram-negative bacteria. Five proteins of Sulfolobus solfataricus, SSO0120, SSO0572, SSO2316, SSO2387 and SSO2680, which are homologous to secretion ATPases of bacterial type II, type IV secretion systems and the type IV pili assembly machinery, were identified. The operon structures of these putative secretion systems encoding gene clusters and the expression patterns of the ATPases under different growth conditions were determined, and it was established that all five putative ATPases do show a divalent cation-dependent ATPase activity at high temperature. These results show that the archaeal secretion systems are related to the bacterial secretion systems and might be powered in a similar way.
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Evidence for two recA genes mediating DNA repair in Bacillus megaterium
More LessIsolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.
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Hypoxia abolishes transience of the heat-shock response in the methylotrophic yeast Hansenula polymorpha
More LessThe heat-shock response is conserved amongst practically all organisms. Almost invariably, the massive heat-shock protein (Hsp) synthesis that it induces is subsequently down-regulated, making this a transient, not a sustained, stress response. This study investigated whether the heat-shock response displays any unusual features in the methylotrophic yeast Hansenula polymorpha, since this organism exhibits the highest growth temperature (49–50 °C) identified to date for any yeast and grows at 47 °C without either thermal death or detriment to final biomass yield. Maximal levels of Hsp induction were observed with a temperature upshift of H. polymorpha from 30 °C to 47–49 °C. This heat shock induces a prolonged growth arrest, heat-shock protein synthesis being down-regulated long before growth resumes at such high temperatures. A 30 °C to 49 °C heat shock also induced thermotolerance, although H. polymorpha cells in balanced growth at 49 °C were intrinsically thermotolerant. Unexpectedly, the normal transience of the H. polymorpha heat-shock response was suppressed completely by imposing the additional stress of hypoxia at the time of the 30 °C to 49 °C temperature upshift. Hypoxia abolishing the transience of the heat-shock response appears to operate at the level of Hsp gene transcription, since the heat-induced Hsp70 mRNA was transiently induced in a heat-shocked normoxic culture but displayed sustained induction in a culture deprived of oxygen at the time of temperature upshift.
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Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region
More LessLasX regulates the transcription of the divergent operons lasXY and lasA–W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA–lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P lasA–W , and a third imperfect copy of this motif, overlapping the −10 element of P lasA–W , were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.
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Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16
More LessPhasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.
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Novel stationary-phase-upregulated protein of Porphyromonas gingivalis influences production of superoxide dismutase, thiol peroxidase and thioredoxin
Porphyromonas gingivalis, an obligately anaerobic bacterium, is implicated as a major pathogen in the development and progression of chronic periodontitis. Although expression of several virulence factors of the bacterium has been found to be affected by environmental stress such as entrance into the stationary growth phase and heat, there is relatively little information on the mechanisms that may operate in the bacterium in response to environmental stress. In this study, a novel protein (UstA) was investigated that was initially identified following two-dimensional gel analysis. Expression of UstA was upregulated in stationary phase or by exposure to atmospheric oxygen. N-terminal sequencing and database analysis with the P. gingivalis genome sequence revealed that the UstA-encoding gene (ustA) was located upstream of a homologue of the usp gene encoding the universal stress protein on the chromosome. The ustA gene appeared to be transcribed in a monocistronic fashion, as revealed by primer extension and Northern blot analysis. To elucidate the role of UstA in the bacterium, chromosomal mutants carrying a disruption of the ustA gene were constructed. The ustA mutant grew slower than the wild-type parent strain in rich medium, resulting in a lower yield in stationary phase. Furthermore, in this mutant, expression levels of the P. gingivalis homologues of superoxide dismutase, thiol peroxidase and thioredoxin were markedly higher than those in the wild-type, especially in stationary phase. The ustA mutant was more resistant to diamide, a thiol-specific oxidant, than the wild-type. In addition, the ustA mutation suppressed hypersensitivities of the oxyR mutant to diamide, metronidazole and mitomycin C. These results suggest that UstA may play a significant role in oxidative stress responses in the bacterium.
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Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant
More LessThe dnaX gene of Escherichia coli encodes τ and γ clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and τ or γ is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.
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- Biodiversity And Evolution
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CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies
More LessThe remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or ‘spacers’. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.
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Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain
Candida albicans strain SC5314 contains two ALS3 alleles, which differ in sequence with respect to the number of copies of the 108 bp tandem repeat sequence within the central domain of the coding region. One allele (ALS3(12)) has 12 tandem repeat copies while the other (ALS3(9)) has 9 copies. Wild-type C. albicans (ALS3(12)/ALS3(9)) and those containing various ALS3 alleles (ALS3(12)/als3Δ(9), als3Δ(12)/ALS3(9) and als3Δ(12)/als3Δ(9)) were assayed for adhesion to monolayers of cultured vascular endothelial and pharyngeal epithelial cells. These assays showed obvious adhesive function for the larger Als3p protein, compared to a minor contribution to adhesion from the smaller protein. These functional differences in strain SC5314 prompted examination of ALS3 allelic diversity across the five major genetic clades of C. albicans. This analysis focused on the number of copies of the tandem repeat sequence within the central domain of the coding region and showed a range of alleles encoding from 6 to 19 tandem repeat copies. Clades differed with respect to prevalent ALS3 alleles and allele distribution, but were similar for the mean number of tandem repeat copies per ALS3 allele. Analysis of allelic pairing showed clade differences and the tendency for C. albicans strains to encode one longer and one shorter ALS3 allele. The allelic variability observed for ALS3 and its functional consequences observed in strain SC5314 highlight the importance of understanding ALS allelic diversity in order to draw accurate conclusions about Als protein function.
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- Environmental Microbiology
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Statistical assessment of a laboratory method for growing biofilms
Microbial biofilms have been grown in laboratories using a variety of different approaches. A laboratory biofilm reactor system, called the CDC biofilm reactor (CBR) system, has been devised for growing biofilms under moderate to high fluid shear stress. The reactor incorporates 24 removable biofilm growth surfaces (coupons) for sampling and analysing the biofilm. Following preliminary experiments to verify the utility of the CBR system for growing biofilms of several clinically relevant organisms, a standard operating procedure for growing a Pseudomonas aeruginosa biofilm was created. This paper presents the results of a rigorous, intra-laboratory, statistical evaluation of the repeatability and ruggedness of that procedure as well as the results of the experiments with clinically relevant organisms. For the statistical evaluations, the outcome of interest was the density (c.f.u. cm−2) of viable P. aeruginosa. Replicate experiments were conducted to assess the repeatability of the log density outcome. The mean P. aeruginosa log10 density was 7·1, independent of the coupon position within the reactor. The repeatability standard deviation of the log density based on one coupon per experiment was 0·59. Analysis of variance showed that the variability of the log density was 53 % attributable to within-experiment sources and 47 % attributable to between-experiments sources. The ruggedness evaluation applied response-surface design and regression analysis techniques, similar to those often used for sensitivity analyses in other fields of science and engineering. This approach provided a quantitative description of ruggedness; specifically, the amount the log density was altered by small adjustments to four key operational factors – time allowed for initial surface colonization, temperature, nutrient concentration, and fluid shear stress on the biofilm. The small size of the regression coefficient associated with each operational factor showed that the method was rugged; that is, relatively insensitive to minor perturbations of the four factors. These results demonstrate that the CBR system is a reliable experimental tool for growing a standard biofilm in the laboratory and that it can be adapted to study several different micro-organisms.
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- Genes And Genomes
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Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans
More LessThe presence of a 174 kb plasmid called pMUM001 in Mycobacterium ulcerans, the first example of a mycobacterial plasmid encoding a virulence determinant, was recently reported. Over half of pMUM001 is devoted to six genes, three of which encode giant polyketide synthases (PKS) that produce mycolactone, an unusual cytotoxic lipid produced by M. ulcerans. In this present study the remaining 75 non-PKS-associated protein-coding sequences (CDS) are analysed and it is shown that pMUM001 is a low-copy-number element with a functional ori that supports replication in Mycobacterium marinum but not in the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Sequence analyses revealed a highly mosaic plasmid gene structure that is reminiscent of other large plasmids. Insertion sequences (IS) and fragments of IS, some previously unreported, are interspersed among functional gene clusters, such as those genes involved in plasmid replication, the synthesis of mycolactone, and a potential phosphorelay signal transduction system. Among the IS present on pMUM001 were multiple copies of the high-copy-number M. ulcerans elements IS2404 and IS2606. No plasmid transfer systems were identified, suggesting that trans-acting factors are required for mobilization. The results presented here provide important insights into this unusual virulence plasmid from an emerging but neglected human pathogen.
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Comparison of gene expression in trap cells and vegetative hyphae of the nematophagous fungus Monacrosporium haptotylum
Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. The global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum were compared. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridized to a cDNA array containing probes of 2822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23·3 % (657 of 2822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Among them were several putative homologues for small GTPases, such as rho1, rac1 and ras1, and a rho GDP dissociation inhibitor (rdi1). Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. In the last category, a glycogen phosphorylase (gph1) gene homologue, one of the most upregulated genes in the knobs as compared to mycelium, was characterized. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant-pathogenic fungi. Among them, a gas1 (mas3) gene homologue (designated gks1), which is specifically expressed in appressoria of the rice blast fungus, was characterized.
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Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240
More LessA single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind l-arginine with high affinity (K d=0·39±0·06 μM). Competition experiments revealed only partial inhibition by excess l-lysine (38 %) and l-ornithine (46 %), while no inhibition was observed with l-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of l-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 °C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0·71 μmol Pi min−1 (mg protein)−1 and a K m (ATP) of 1·59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.
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Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons
More LessThe combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2·061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (∼0·05–4·3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA–D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S–23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.
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The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents
More LessA 7·5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1–L3), ten trachoma isolates (serovars A–C) and nine urogenital isolates (serovars D–J). The results indicated an average plasmid copy number of 4·0±0·8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7·6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7·4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1·3±0·2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.
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Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1
More LessThe sequence of 50 625 bp of chromosomal DNA derived from Shiga-toxin (Stx)-producing Escherichia coli (STEC) O111 : H− strain 1639/77 was determined. This DNA fragment contains the cryptic Stx1-encoding prophage CP-1639 and its flanking chromosomal regions. The genome of CP-1639 basically resembles that of lambdoid phages in structure, but contains three IS629 elements, one of which disrupts the gene of a tail fibre component. The prophage genome lacks parts of the recombination region including integrase and excisionase genes. Moreover, a capsid protein gene is absent. CP-1639 is closely associated with an integrase gene of an ancient integrative element. This element consists of three ORFs of unknown origin and a truncated integrase gene homologous to intA of CP4-57. By PCR analysis and sequencing, it was shown that this integrative element is present in a number of non-O157 STEC serotypes and in non-STEC strains, where it is located at the 3′-end of the chromosomal ssrA gene. Whereas in most E. coli O111 : H− strains, prophages are inserted in this site, E. coli O26 strains contain the integrative element not connected to a prophage. In E. coli O103 strains, the genetic structure of this region is variable. Comparison of DNA sequences of this particular site in E. coli O157 : H7 strain EDL933, E. coli O111 : H− strain 1639/77 and E. coli K-12 strain MG1655 showed that the ssrA gene is associated in all cases with the presence of foreign DNA. The results of this study have shown that the cryptic prophage CP-1639 is associated with an integrative element at a particular site in the E. coli chromosome that possesses high genetic variability.
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The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation
More LessPseudomonas aeruginosa is a Gram-negative bacterium associated with nosocomial infections and cystic fibrosis. Chronic bacterial infections are increasingly associated with the biofilm lifestyle in which microcolonies are embedded in an extracellular matrix. Screening procedures for identifying biofilm-deficient strains have allowed the characterization of several key determinants involved in this process. Biofilm-deficient P. aeruginosa PAK strains affected in a seven-gene cluster called pel were characterized. The pel genes encode proteins with similarity to components involved in polysaccharide biogenesis, of which PelF is a putative glycosyltransferase. PelG was also identified as a putative component of the polysaccharide transporter (PST) family. The pel genes were previously identified in the P. aeruginosa PA14 strain as required for the production of a glucose-rich matrix material involved in the formation of a thick pellicle and resistant biofilm. However, in PA14, the pel mutants have no clear phenotype in the initiation phase of attachment. It was shown that pel mutations in the PAK strain had little influence on biofilm initiation but, as in PA14, appeared to generate the least robust and mature biofilms. Strikingly, by constructing pel mutants in a non-piliated P. aeruginosa PAK strain, an unexpected effect of the pel mutation in the early phase of biofilm formation was discovered, since it was observed that these mutants were severely defective in the attachment process on solid surfaces. The pel gene cluster is conserved in other Gram-negative bacteria, and mutation in a Ralstonia solanacearum pelG homologue, ragG, led to an adherence defect.
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- Pathogens And Pathogenicity
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Identification of the outer-membrane protein PagC required for the serum resistance phenotype in Salmonella enterica serovar Choleraesuis
More LessSerum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.
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Diverse roles for HspR in Campylobacter jejuni revealed by the proteome, transcriptome and phenotypic characterization of an hspR mutant
Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of hspR in C. jejuni, members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the hspR mutant, including the chaperone genes dnaK, grpE and clpB, and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the hspR mutant was unable to form colonies at 44 °C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the hspR mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for hspR mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of hspR also reduced the ability of Campylobacter to adhere to and invade human epithelial INT-407 cells in vitro, possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in hspR mutant cells. It was concluded that, in C. jejuni, HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.
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Phase variation mediated niche adaptation during prolonged experimental murine infection with Helicobacter pylori
More LessChanges in the repeats associated with the recently redefined repertoire of 31 phase-variable genes in Helicobacter pylori were investigated following murine gastric colonization for up to one year in three unrelated H. pylori strains. Between the beginning and end of the experimental period, changes were seen in ten genes (32 %), which would alter gene expression in one or more of the three strains studied. For those genes that showed repeat length changes at the longest time points, intermediate time points showed differences between the rates of change for different functional groups of genes. Genes most likely to be associated with immediate niche fitting changed most rapidly, including phospholipase A (pldA) and LPS biosynthetic genes. Other surface proteins, which may be under adaptive immune selection, changed more slowly. Restriction-modification genes showed no particular temporal pattern. The number of genes that phase varied during adaptation to the murine gastric environment correlated inversely with their relative fitness as previously determined in this murine model of colonization. This suggests a role for these genes in determining initial fitness for colonization as well as in subsequent niche adaptation. In addition, a coding tandem repeat within a phase-variable gene which does not control actual gene expression was also investigated. This repeat was found to vary in copy number during colonization. This suggests that changes in the structures encoded by tandem repeats may also play a role in altered protein functions and/or immune evasion during H. pylori colonization.
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The Dps-like protein Fri of Listeria monocytogenes promotes stress tolerance and intracellular multiplication in macrophage-like cells
Members of the ferritin-like Dps protein family are found in a number of bacterial species, where they demonstrate the potential to bind iron, and have been implicated in tolerance to oxidative stress. In this study of the food-borne pathogen Listeria monocytogenes, the fri gene encoding a Dps homologue was deleted, and, compared to wild-type cells, it was found that the resulting mutant was less resistant to hydrogen peroxide, and demonstrated reduced survival following long-term (7–11 days) incubation in laboratory media. In view of this, it is shown that fri gene expression is controlled by the hydrogen peroxide regulator PerR, as well as the general stress sigma factor σ B. When fri mutant cells were transferred to iron-limiting conditions, growth was retarded relative to wild-type cells, indicating that Fri may be required for iron storage. This notion is supported by the observation that the L. monocytogenes genome appears not to encode other ferritin-like proteins. Given the role of Fri in resistance to oxidative stress, and growth under iron-limiting conditions, the ability of the fri mutant to infect mice was examined. When injected by the intraperitoneal route, the fri mutant demonstrated a reduced capacity to proliferate in the organs of infected mice relative to the wild-type, whereas when the bacteria were supplied intravenously this effect was mitigated. In addition, the mutant was impaired in its ability to survive and grow in J774.A1 mouse macrophage cells. Thus, the data suggest that Fri contributes to the ability of L. monocytogenes to survive in environments where oxidative stress and low iron availability may impede bacterial proliferation.
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The vlhA loci of Mycoplasma synoviae are confined to a restricted region of the genome
More LessMycoplasma synoviae, a major pathogen of poultry, contains a single expressed, full-length vlhA gene encoding its haemagglutinin, and a large number of vlhA pseudogenes that can be recruited by multiple site-specific recombination events to generate chimaeric variants of the expressed gene. The position and distribution of the vlhA pseudogene regions, and their relationship with the expressed gene, have not been investigated. To determine the relationship between these regions, a physical map of the M. synoviae genome was constructed using the restriction endonucleases SmaI, I-CeuI, BsiWI, ApaI and XhoI and radiolabelled probes for rrnA, recA and tufA. A cloned fragment encoding the unique portion of the expressed vlhA gene and two PCR products containing conserved regions of the ORF 3 and ORF 6 vlhA pseudogenes were used to locate the regions containing these genes on the map. The chromosome of M. synoviae was found to be 890·4 kb and the two rRNA operons were in the same orientation. Both the expressed vlhA gene and the vlhA pseudogenes were confined to the same 114 kb region of the chromosome. These findings indicate that, unlike Mycoplasma gallisepticum, in which the vlhA genes are located in several loci around the chromosome and in which antigenic variation is generated by alternating transcription of over 40 translationally competent genes, M. synoviae has all of the vlhA sequences clustered together, suggesting that close proximity is needed to facilitate the site-specific recombinations used to generate diversity in the expressed vlhA gene.
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Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri
Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 °C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 °C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 °C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.
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- Physiology
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Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone
More LessMycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.
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Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state
More LessThe Escherichia coli K-12 strain TG1 was grown at 28 °C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0·044 to 0·415 h−1. The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0·3 h−1. At dilution rates between 0·3 h−1 and 0·4 h−1, a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0·4 h−1. An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0·4 h−1, close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.
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Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour
More LessThe metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, d- and l-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached. In the downshift experiment, the adenylate energy charge behaved inversely; no by-products were formed, indicating a tight coupling of anabolism and catabolism. Both dilution rate shifts were accompanied by an instantaneous increase of cAMP, presaging the subsequent changes in metabolic pathway utilization. Intracellular key metabolites of the Embden–Meyerhof–Parnas (EMP) pathway were measured to evaluate the metabolic perturbation during the upshift. Fructose 1,6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP) increased rapidly after the upshift, while glyceraldehyde 3-phosphate decreased. It is concluded that this imbalance at the branch-point of FDP induces the methylglyoxal (MG) pathway, a low-energy-yielding bypass of the lower EMP pathway, through the increasing level of DHAP. MG pathway activation after the upshift was simulated by restricting anabolic rates using a stoichiometry-based metabolic model. The metabolic model predicted that low-energy-yielding catabolic pathways are utilized preferentially in the transient after the upshift. Upon severe dilution rate upshifts, an oscillatory behaviour occurred, apparent through long-term oscillations of respiratory activity, which started when the cytotoxic compound MG reached a threshold concentration of 1·5 mg l−1 in the medium.
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Influence of flavomycin on ruminal fermentation and microbial populations in sheep
More LessFlavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin day−1 decreased ruminal ammonia and total volatile fatty acid concentrations (P<0·001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some high-activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.
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The pimFABCDE operon from Rhodopseudomonas palustris mediates dicarboxylic acid degradation and participates in anaerobic benzoate degradation
More LessBacteria in anoxic environments typically convert aromatic compounds derived from pollutants or green plants to benzoyl-CoA, and then to the C7 dicarboxylic acid derivative 3-hydroxypimelyl-CoA. Inspection of the recently completed genome sequence of the purple nonsulfur phototroph Rhodopseudomonas palustris revealed one predicted cluster of genes for the β-oxidation of dicarboxylic acids. These genes, annotated as pimFABCDE, are predicted to encode acyl-CoA ligase, enoyl-CoA hydratase, acyl-CoA dehydrogenase and acyl-CoA transferase enzymes, which should allow the conversion of odd-chain dicarboxylic acids to glutaryl-CoA, and even-chain dicarboxylic acids to succinyl-CoA. A mutant strain that was deleted in the pim gene cluster grew at about half the rate of the wild-type parent when benzoate or pimelate was supplied as the sole carbon source. The mutant grew five times more slowly than the wild-type on the C14 dicarboxylic acid tetradecanedioate. The mutant was unimpaired in growth on the C8-fatty acid caprylate. The acyl-CoA ligase predicted to be encoded by the pimA gene was purified, and found to be active with C7–C14 dicarboxylic and fatty acids. The expression of a pimA–lacZ chromosomal gene fusion increased twofold when cells were grown in the presence of straight-chain C7–C14 dicarboxylic and fatty acids. These results suggest that the β-oxidation enzymes encoded by the pim gene cluster are active with medium-chain-length dicarboxylic acids, including pimelate. However, the finding that the pim operon deletion mutant is still able to grow on dicarboxylic acids, albeit at a slower rate, indicates that R. palustris has additional genes that can also specify the degradation of these compounds.
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- Plant-Microbe Interactions
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Two site-specific recombinases are implicated in phenotypic variation and competitive rhizosphere colonization in Pseudomonas fluorescens
The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the sss gene. Here, it is shown that a second recombinase encoded by the xerD gene is also implicated in phenotypic variation. A putative xerD gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the λ integrase family. Mutants affected in the sss or xerD genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the sss gene suppressed the phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on the phenotype of an sss mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the gacA/S genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the sss and xerD genes with a promoterless lacZ gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.
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- Theoretical Microbiology
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Skew-Laplace distribution in Gram-negative bacterial axenic cultures: new insights into intrinsic cellular heterogeneity
More LessThe application of flow cytometry and skew-Laplace statistical analysis to assess cellular heterogeneity in Gram-negative axenic cultures is reported. In particular, fit to the log-skew-Laplace distribution for cellular side scatter or ‘granulosity’ is reported, and a number of theoretical and applied issues are considered in relation to the biological significance of this fit.
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