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Volume 152,
Issue 3,
2006
Volume 152, Issue 3, 2006
- Mini-Review
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From genomes to function: haloarchaea as model organisms
More LessHaloarchaea are adapted to high-salt environments and accumulate equally high salt concentrations in the cytoplasm. The genomes of representatives of six haloarchaeal genera have been fully or partially sequenced, allowing the analysis of haloarchaeal properties in silico. Transcriptome and proteome analyses have been established for Halobacterium salinarum and Haloferax volcanii. Genetic systems are available including methods that allow the fast in-frame deletion or modification of chromosomal genes. The high-efficiency transformation system of Hf. volcanii allows the isolation of genes essential for a biological process by complementation of loss-of-function mutants. For the analysis of haloarchaeal biology many molecular genetic, biochemical, structural and cell biological methods have been adapted to application at high salt concentrations. Recently it has become clear that several different mechanisms allow the adaptation of proteins to the high salt concentration of the cytoplasm. Taken together, the wealth of techniques available make haloarchaea excellent archaeal model species.
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- Cell And Developmental Microbiology
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The Rho3 and Rho4 small GTPases interact functionally with Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae
Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1Δwsc1Δ synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1Δ cells. Inactivation of RHO3 or RHO4 was able to rescue the rgd1Δwsc1Δ synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC–mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC–MAP kinase pathway.
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- Biochemistry And Molecular Biology
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Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans
More LessFludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Δ mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Δ mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Δ MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.
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Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity
More LessThe importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1·2 M the growth curves were similar to those of its wild-type control, at 1·5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.
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Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin
More LessThe gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca2+ requirement and the optimum temperature and pH were 65 °C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.
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RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase
More LessAs in other bacteria, 3′-tails are added post-transcriptionally to Streptomyces coelicolor RNA. These tails are heteropolymeric, and although there are several candidates, the enzyme responsible for their synthesis has not been definitively identified. This paper reports on three candidates for this role. First, it is confirmed that the product of S. coelicolor gene SCO3896, although it bears significant sequence similarity to Escherichia coli poly(A) polymerase I, is a tRNA nucleotidyltransferase, not a poly(A) polymerase. It is further shown that SCO2904 encodes an RNase PH homologue that possesses the polymerization and phosphorolysis activities expected for enzymes of that family. S. coelicolor RNase PH can add poly(A) tails to a model RNA transcript in vitro. However, disruption of the RNase PH gene has no effect on RNA 3′-tail length or composition in S. coelicolor; thus, RNase PH does not function as the RNA 3′-polyribonucleotide polymerase [poly(A) polymerase] in that organism. These results strongly suggest that the enzyme responsible for RNA 3′-tail synthesis in S. coelicolor and other streptomycetes is polynucleotide phosphorylase (PNPase). Moreover, this study shows that both PNPase and the product of SCO3896 are essential. It is possible that the dual functions of PNPase in the synthesis and degradation of RNA 3′-tails make it indispensable in Streptomyces.
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The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501
The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P tra , which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. β-Galactosidase assays with P tra–lacZ fusions proved P tra promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.
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A putative sensor kinase, Hik31, is involved in the response of Synechocystis sp. strain PCC 6803 to the presence of glucose
The reason(s) for glucose sensitivity in certain cyanobacterial strains is poorly understood. Inactivation of genes encoding the putative sensor kinase Hik31 in Synechocystis sp. strain PCC 6803 resulted in a mutant unable to grow in the presence of d-glucose. Sensitivities to d-glucose, its analogue 2-deoxy-d-glucose, and fructose, were alleviated in mutants in which glcP, encoding the glucose transporter, was inactivated. These data indicate that permeation of these substrates is required to inflict cell death. The mutant Δhik31, and the glucose-sensitive strain of Synechocystis, do not possess glucokinase activity, although a transcript originating from glk, encoding glucokinase, is present. Inactivation of glk led to severe sensitivity to glucose, indicating that the presence of glucose itself, within the cells, inflicted this sensitivity. On the other hand, sensitivity to 2-deoxy-d-glucose was lower in Δglk, thus distinguishing between the effect of glucose itself and that of its analogue, which, in the absence of glucokinase activity, may not be phosphorylated. Addition of glucose led to a small rise in glucose-6-phosphate dehydrogenase activity in the wild type, but constitutive activity was observed in the Δhik31 mutant regardless of the presence of glucose. Microarray analyses showed only small changes in the abundance of global transcripts in Synechocystis following glucose addition, but the transcription levels of several genes, including icfG, but not glk, were strongly affected by inactivation of hik31. The mechanism(s) whereby Hik31 is involved in glucose sensing and response is discussed.
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Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC
More LessThis study shows that lipid A of Escherichia coli O157 : H7 differs from that of E. coli K-12 in that it has a phosphoform at the C-1 position, which is distinctively modified by a phosphoethanolamine (PEtN) moiety, in addition to the diphosphoryl form. The pmrC gene responsible for the addition of PEtN to the lipid A of E. coli O157 : H7 was inactivated and the changes in lipid A profiles were assessed. The pmrC null mutant still produced PEtN-modified lipid A species, albeit in a reduced amount, indicating that PmrC was not the only enzyme that could be used to add PEtN to lipid A. Natural PEtN substitution was shown to be present in the lipid A of other serotypes of enterohaemorrhagic E. coli and absent from the lipid A of E. coli K-12. However, the cloned pmrC O157 gene in a high-copy-number plasmid generated a large amount of PEtN-substituted lipid A species in E. coli K-12. The occurrence of PEtN-substituted lipid A species was associated with a slight increase in the MICs of cationic peptide antibiotics, suggesting that the lipid A modification with PEtN would be beneficial for survival of E. coli O157 : H7 in certain environmental niches. However, PEtN substitution in the lipid A profiles was not detected when putative inner-membrane proteins (YhbX/YbiP/YijP/Ecf3) that show significant similarity with PmrC in amino acid sequence were expressed from high-copy-number plasmids in E. coli K-12. This suggests that these potential homologues are not responsible for the addition of PEtN to lipid A in the pmrC mutant of E. coli O157 : H7. When cells were treated with EDTA, the amount of palmitoylated lipid A from the cells carrying a high-copy-number plasmid clone of pmrC O157 that resulted in significant increase of PEtN substitution was unchanged compared with cells without PEtN substitution, suggesting that the PEtN moiety substituted in lipid A does not compensate for the loss of divalent cations required for bridging neighbouring lipid A molecules.
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In vivo characterization of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea
More LessIn vivo reconstitution of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea was achieved by expression of the genes in Escherichia coli. LC/MS/MS analysis of the dTDP-sugar intermediates produced by operons containing different sets of genes showed that production of dTDP-d-desosamine from dtdp-4-keto-6-deoxy-d-glucose requires only four biosynthetic steps, catalysed by MegCIV, MegCV, MegDII and MegDIII, and that MegCII is not involved. Instead, bioconversion studies demonstrated that MegCII is needed together with MegCIII to catalyse transfer of d-desosamine to 3-α-mycarosylerythronolide B.
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- Biodiversity And Evolution
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Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes
The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil ( Jospeh et al., Appl Environ Microbiol 69, 7210–7215, 2003 ). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.
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Genetic and phenotypic characterization of Listeria monocytogenes lineage III
Listeria monocytogenes has been previously grouped into three evolutionary groups, termed lineages I, II and III. While lineages I and II are commonly isolated from various sources, lineage III isolates are rare and have several atypical and unique phenotypic characteristics. Relative to their prevalence in other sources, lineage III strains are overrepresented among isolates from food-production animals, and underrepresented among isolates from human clinical cases and foods. This work describes an extensive genotypic and phenotypic characterization of 46 lineage III isolates. Phylogenetic analyses of partial sigB and actA sequences showed that lineage III represents three distinct subgroups, which were termed IIIA, IIIB and IIIC. Each of these lineage III subgroups is characterized by differentiating genotypic and phenotypic characteristics. Unlike typical L. monocytogenes, all subgroup IIIB and IIIC isolates lack the ability to ferment rhamnose. While all IIIC and most IIIB isolates carry the putative virulence gene lmaA, the majority of subgroup IIIA isolates lack this gene. All three lineage III subgroups contain isolates from human clinical cases as well as isolates that are cytopathogenic in a cell culture plaque assay, indicating that lineage III isolates have the potential to cause human disease. The identification of specific genotypic and phenotypic characteristics among the three lineage III subgroups suggests that these subgroups may occupy different ecological niches and, therefore, may be transmitted by different pathways.
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- Environmental Microbiology
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Hg(II) sequestration and protection by the MerR metal-binding domain (MBD)
More LessMerR, the metalloregulator of the bacterial mercury resistance (mer) operon, binds Hg(II) with high affinity. To study the mechanism of metal-induced activation, a small protein was previously engineered embodying in a single polypeptide the metal-binding domain (MBD) ordinarily formed between two monomers of MerR. Here the physiological and biochemical properties of MBD expressed on the cell surface or in the cytosol were examined, to better understand the environments in which specific metal binding can occur with this small derivative. Over 20 000 surface copies of MBD were expressed per Escherichia coli cell, with metal stoichiometries of ∼1·0 Hg(II) per MBD monomer. Cells expressing MBD on their surface in rich medium bound 6·1-fold more Hg(II) than those not expressing MBD. Although in nature cells use the entire mer operon to detoxify mercury, it was interesting to note that cells expressing only MBD survived Hg(II) challenge and recovered more quickly than cells without MBD. Cell-surface-expressed MBD bound Hg(II) preferentially even in the presence of a 22-fold molar excess of Zn(II) and when exposed to equimolar Cd(II) in addition. MBD expressed in the cystosol also afforded improved survival from Hg(II) exposure for E. coli and for the completely unrelated bacterium Deinococcus radiodurans.
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AggA is required for aggregation and increased biofilm formation of a hyper-aggregating mutant of Shewanella oneidensis MR-1
Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type.
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Enzymic systems proposed to be involved in the dissimilatory reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus
More LessVarious enzymic systems, such as nitrite reductase, sulfite reductase and glutathione reductase, have been proposed for, or suspected to be involved in, the reduction of selenite in bacteria. As alphaproteobacteria have been shown to be highly tolerant to transition metal oxyanions, it seemed interesting to investigate the hypothetical involvement of these different enzymes in the reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus. The hypothetical involvement of nitrite reductase and sulfite reductase in the reduction of selenite in these bacteria was investigated by analysing the effects of nitrite and sulfite amendments on the growth and kinetics of selenite reduction. The reduction of selenite was not concomitant with that of either sulfite or nitrite in Rs. rubrum, suggesting that the reduction pathways operate independently. In Rb. capsulatus, strong interactions were observed between the nitrite reduction and selenite reduction pathways. However, in both organisms, selenite reduction took place during both the growth phase and the stationary phase, indicating that selenite metabolism is constitutively expressed. In contrast, neither nitrite nor sulfite was transformed during stationary phase, suggesting that the metabolism of both ions is induced, which implies that identical reduction pathways for selenite and nitrite or selenite and sulfite are excluded. Buthionine sulfoximine (BSO, S-n-butyl homocysteine sulfoximine), a specific inhibitor of glutathione synthesis, was used to depress the intracellular glutathione level. In stationary-phase cultures of both Rs. rubrum and Rb. capsulatus amended with BSO, the rate of reduction of selenite was slowed, indicating that glutathione may be involved in the dissimilatory reduction of selenite in these organisms. The analysis of the headspace gases of the cultures indicated that the synthesis of methylated selenium compounds was prevented in the presence of 3·0 mM BSO in both organisms, implying that glutathione is also involved in the transformation of selenite to volatile selenium compounds.
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- Genes And Genomes
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A SitABCD homologue from an avian pathogenic Escherichia coli strain mediates transport of iron and manganese and resistance to hydrogen peroxide
More LessAn operon encoding a member of the family of ATP-binding cassette (ABC) divalent metal ion transporters, homologous to Salmonella enterica SitABCD, has been identified in the avian pathogenic Escherichia coli (APEC) strain χ7122. The sitABCD genes were located on the virulence plasmid pAPEC-1, and were highly similar at the nucleotide level to the chromosomally encoded sitABCD genes present in Shigella spp. A cloned copy of sitABCD conferred increased growth upon a siderophore-deficient E. coli strain grown in nutrient broth supplemented with the chelator 2,2′-dipyridyl. Ion rescue demonstrated that Sit-mediated growth promotion of this strain was due to the transport of iron. SitABCD mediated increased transport of both iron and manganese as demonstrated by uptake of 55Fe, 59Fe or 54Mn in E. coli K-12 strains deficient for the transport of iron (aroB feoB) and manganese (mntH) respectively. Isotope uptake and transport inhibition studies showed that in the iron transport deficient strain, SitABCD demonstrated a greater affinity for iron than for manganese, and SitABCD-mediated transport was higher for ferrous iron, whereas in the manganese transport deficient strain, SitABCD demonstrated greater affinity for manganese than for iron. Introduction of the APEC sitABCD genes into an E. coli K-12 mntH mutant also conferred increased resistance to the bactericidal effects of hydrogen peroxide. APEC strain χ7122 derivatives lacking either a functional SitABCD or a functional MntH transport system were as resistant to hydrogen peroxide as the wild-type strain, whereas a Δsit ΔmntH double mutant was more sensitive to hydrogen peroxide. Overall, the results demonstrate that in E. coli SitABCD represents a manganese and iron transporter that, in combination with other ion transport systems, may contribute to acquisition of iron and manganese, and resistance to oxidative stress.
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- Genome Update
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- Pathogens And Pathogenicity
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Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR
More LessProduction of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 °C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 °C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 °C. However, incubation at 44 °C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 °C was evident.
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Genetic diversity of the C protein β-antigen gene and its upstream regions within clonally related groups of type Ia and Ib group B streptococci
More LessC protein β antigen (Bac), a surface protein of group B streptococci (GBS), is known to concurrently bind the Fc portion of IgA and factor H (FH). The authors' previous work has demonstrated that mRNA expression levels show diversity among clonally related strains containing genes (bac) encoding Bac, with high expression noted in invasive strains. In this study, the bac gene and upstream regions containing putative promoters, three ORFs and an IS1381 insertion sequence were characterized. Three invasive strains showed high bac expression levels and did not show any notable mutations except one strain producing Bac that was able to bind FH but not IgA. A deletion of 51 amino acid residues, including part of the Bac IgA-binding region, was identified and hypothesized to contribute to the loss of the IgA-binding ability of this strain. A vaginal strain that showed somewhat higher bac expression levels and produced Bac lacking immunoreactivity contained an 11 bp deletion, which generated a premature termination codon, in the region preceding the IgA-binding region. In another vaginal strain that did not express bac, disruption of the upstream ORFs of the sensor histidine kinase and DNA-binding response regulator, due to frameshift mutations, was noted although it is not known whether these proteins directly affect bac expression levels. An IS1381 insertion into the promoter region was found in another vaginal strain that showed low expression levels and produced Bac with a significantly larger proline-rich repeat region. These results demonstrate considerable genetic diversity of the bac and upstream regions of invasive and noninvasive GBS, which may contribute to the variability of bac expression levels among those strains.
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The encapsulation of enterotoxigenic Escherichia coli colonization factor CS3 in biodegradable microspheres enhances the murine antibody response following intranasal administration
More LessThe aim of this study was to measure serum and mucosal antibody responses following intranasal administration of biodegradable poly(dl-lactide-co-glycolide) (PLGA) microspheres loaded with the CS3 colonization factor isolated from enterotoxigenic Escherichia coli (ETEC). The response was compared against that measured in mice similarly administered the native CS3 antigen and in mice co-administered, along with the CS3 antigen, a known mucosal adjuvant, the R192G mutant heat-labile enterotoxin (mLT). The integrity of the CS3 antigen released from the microspheres was maintained as determined by SDS-PAGE and immunoblotting. Native CS3 induced serum and mucosal (bronchoalveolar, small intestinal and faecal) IgG and IgA responses. The co-administration of the mLT mucosal adjuvant significantly enhanced (P<0·001) serum and mucosal antibody responses to the CS3 protein. Likewise, the CS3-loaded PLGA microspheres induced significantly greater (P<0·001) serum and mucosal antibody responses than native CS3, as well as inducing antibody responses superior to those of the CS3 plus mLT formulation. Following administration of CS3 plus mLT, the mice became distressed (loss of activity, increased huddling, ruffled fur), a situation not seen following administration of the CS3-loaded PLGA microspheres. The results in this trial show that the CS3-loaded PLGA microspheres when administered intranasally to mice caused no observable distress to the mice and significantly (P<0·001) enhanced the immunogenicity of the CS3 protein.
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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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)
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