- Volume 148, Issue 1, 2002
Volume 148, Issue 1, 2002
- Mini-Review
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- Sgm Special Lecture
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- Microbiology Comment
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- Research Paper
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Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator
More LessHigh-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities: an externally directed surface ferric reductase, a membrane-associated PPD (p-phenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.
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Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis
Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l−1. N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)–Harvey-RasVal12 fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have potential for industrial production of soluble and active scFv.
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The candicidin gene cluster from Streptomyces griseus IMRU 3570
More LessThe GenBank accession numbers for the sequences reported in this paper are AJ300302 and AJ300303.
A 205 kb DNA region from Streptomyces griseus IMRU 3570, including the candicidin biosynthetic gene cluster, was cloned and partially sequenced. Analysis of the sequenced DNA led to identification of genes encoding part of a modular polyketide synthase (PKS), genes for thioesterase, macrolactone ring modification, mycosamine biosynthesis and attachment to the macrolide ring, candicidin export and regulatory proteins. It represents the first extensive genetic characterization of an aromatic polyene macrolide antibiotic biosynthetic gene cluster. Of particular interest is the presence of the CanP1 loading domain (the first described as responsible for the activation of an aromatic starter unit) and the polypeptide CanP3 (carrying modules for the formation of five out of seven conjugated double bonds). Disruption of the pabAB gene that encodes the starter unit of candicidin abolished its production [which was restored when exogenous p-aminobenzoic acid (PABA) was supplied to the culture] and resulted in an enhanced production of another antifungal compound that is barely detected in the wild-type.
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Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens
More LesspSAM2 is integrated into the Streptomyces ambofaciens chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) site. The 43 kDa integrase protein encoded by pSAM2 catalyses this recombination event. Tools have been developed to study site-specific recombination in Escherichia coli. In vivo studies showed that a 360 bp fragment of attP is required for efficient site-specific recombination and that int can be provided in trans. pSAM2 integrase was purified and overexpressed in E. coli and Int binding at the attP site was studied. DNaseI footprinting revealed two sites that bind integrase strongly and appear to be symmetrical with regard to the core site. These two P1/P2 arm-type sites both contain a 17 bp motif that is identical except at one position, GTCACGCAG(A/T)TAGACAC. P1 and P2 are essential for site-specific recombination.
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Investigation of in vivo cross-talk between key two-component systems of Escherichia coli
More LessIntracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.
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Fast lysis of Escherichia coli filament cells requires differentiation of potential division sites
More LessPeriodic activation of zonal peptidoglycan (murein) synthesis at division sites in Escherichia coli has been reported recently. Zonal synthesis is responsible for septum formation, whereas elongation of the cell sacculus is performed by diffuse insertion of precursors. Zonal synthesis can be triggered in ftsA, ftsQ and ftsI (pbpB) division mutants growing as filaments at the restrictive temperature, but not in ftsZ mutant strains. The lytic response to β-lactams of cells able or unable to periodically trigger a zonal mode of murein synthesis could be substantially different. Therefore, we investigated the response to the bacteriolytic β-lactam cefsulodin of ftsZ and ftsI mutants growing at the restrictive (42 °C) temperature. The ftsI cells lysed early and quickly after addition of the antibiotic. Sacculi of lysed cells were transversely cut in a very sharp way. In contrast the ftsZ strain lysed late and slowly after addition of the antibiotic and sacculi showed a generalized weakening of the murein network and extended breaks with a frayed appearance. No transversely cut sacculi comparable to those seen in the ftsI samples were found. Our results strongly support that β-lactam-induced lysis occurs preferentially at division sites because of the activation of zonal murein synthesis at the initiation of septation.
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Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound
Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive, single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra, is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.
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kdsA mutations affect FtsZ-ring formation in Escherichia coli K-12
No one has, as yet, addressed the relationship between the nature of the outer membrane and cell division. kdsA encodes 3-deoxy-D-manno-octulosonic acid (KDO) 8-phosphate synthetase which catalyses the first step in the synthesis of KDO, the linker between lipid A and oligosaccharide of lipopolysaccharide (LPS). Seven temperature-sensitive mutants containing missense mutations in kdsA were affected in the production of KDO and all mutants stopped dividing at 41 °C and formed filaments with either one or no FtsZ ring. All observed defects were reversed by the plasmid-borne wild-type kdsA gene. Western blotting analysis, however, demonstrated that the amount of FtsZ protein was not affected by the mutation. The mutants were more susceptible to various hydrophobic materials, such as novobiocin, eosin Y and SDS at 36 °C. Methylene blue, however, restored kdsA mutant growth. Plasmid-borne wild-type msbA, encoding a lipid A transporter in the ABC family, partially suppressed kdsA mutation. A mutation of lpxA, functioning at the first stage in lipid A biosynthesis, inhibited both cell division and growth, producing short filaments. These results indicate that the instability of the outer membrane, caused by the defect in KDO biosynthesis, affects FtsZ-ring formation.
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The starvation-stress response of Salmonella enterica serovar Typhimurium requires σE-, but not CpxR-regulated extracytoplasmic functions
More LessStarvation of Salmonella enterica serovar Typhimurium (S. Typhimurium) for an exogenous source of carbon and energy (C-starvation) induces the starvation-stress response (SSR). The SSR functions to (i) maintain viability during long-term C-starvation and (ii) generate cross-resistance to other environmental stresses. The SSR is, at least partially, under the control of the alternative sigma factor, σS. It is hypothesized that C-starvation causes cell envelope stresses that could induce the σE and/or Cpx regulons, both of which control extracytoplasmic functions and, thus, may play a role in the regulation of the SSR. In support of this hypothesis, Western blot analysis showed that the relative levels of σE increased during C-starvation, peaking after approximately 72 h of C-starvation; in contrast, CpxR levels remained relatively constant from exponential phase up to 72 h of C-starvation. To determine if σE, and thus the regulon it controls, is an essential component of the SSR, several mutant strains were compared for their abilities to survive long-term C-starvation and to develop C-starvation-induced (CSI) cross-resistances. An rpoE mutant strain was significantly impaired in both long-term C-starvation survival (LT-CSS) and in CSI cross-resistance to challenges with 20 mM H2O2 for 40 min, 55 °C for 16 min, pH 3·1 for 60 min and 870·2 USP U polymyxin B ml−1 (PmB) for 60 min, to varying degrees. These results suggest that C-starvation can generate signals that induce the rpoE regulon and that one or more members of the σE regulon are required for maximal SSR function. Furthermore, evidence suggests that the σE and σS regulons function through separate mechanisms in the SSR. In contrast, C-starvation does not appear to generate signals required for Cpx regulon induction which support the findings that it is not required for LT-CSS or cross-resistance to H2O2, pH 3·1 or PmB challenges. However, it was required to achieve maximal cross-resistance to 55 °C. Therefore, σE is a key regulatory component of the SSR and represents an additional σ factor required for the SSR of Salmonella.
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Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium
Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.
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AcnC of Escherichia coli is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates
More LessEscherichia coli possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by acnAB double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the prpD gene of the propionate catabolic operon (prpRBCDE) is identical to AcnC. Inactivation of prpD abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of ybhJ, an unidentified acnA paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate cis-aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron–sulphur protein (M r 54000) having one unstable [2Fe–2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe2+ under reducing conditions.
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Fur-mediated transcriptional and post-transcriptional regulation of FeSOD expression in Escherichia coli
More LessFur (ferric uptake regulation protein) activates sodB expression, increasing expression levels by a factor of seven and sodB transcript stability by a factor of three. Post-transcriptional regulation of sodB was investigated by searching for endoribonucleases that might be involved in sodB mRNA degradation. The activation of sodB expression was significantly reduced if both the RNaseE and RNaseIII genes were mutated. This correlated with cleavage at a palindromic sequence located in the 5′ untranslated region of the sodB transcript. An RNA-binding assay showed that Fur did not directly protect the sodB transcript. It was hypothesized that the persistence of Fur-mediated activation of sodB expression in the RNase double mutant was probably due to an effect at the transcriptional level. Therefore, it was investigated whether Fur had a direct transcriptional effect in vitro. Fur bound the sodB promoter region with low affinity, but it was not able to increase sodB transcription. H-NS-mediated repression of sodB expression, which has been shown to be Fur-dependent, was characterized. No DNA-bending region was identified in the sodB promoter region. H-NS did not interfere with the post-transcriptional effect of Fur. Fur-dependent H-NS and the Fur post-transcriptional effect were not additive. This suggests that Fur and H-NS effects are indirect and may be mediated by a common intermediate.
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Immunodominant membrane proteins from two phytoplasmas in the aster yellows clade (chlorante aster yellows and clover phyllody) are highly divergent in the major hydrophilic region
More LessThe GenBank accession numbers for the sequences reported in this paper are AF244540 and AF244541 for AY-C and CP, respectively.
The mechanisms by which phytoplasmas interact with their hosts are not understood. Mollicute membrane proteins may play a role in such interactions and therefore the amp genes encoding immunodominant proteins from two phytoplasmas, aster yellows and clover phyllody, which fall within the largest taxonomic subclade of the phytoplasmas, have been cloned and characterized. The putative translation products, antigenic membrane proteins (Amps), of these genes have properties which are typical for bacterial membrane proteins, and which suggest that each has a single large extracellular hydrophilic domain held by a transmembrane region near the C-terminus, with only a short C-terminal intracellular sequence. Both of the Amps characterized here have bacterial leader sequences which are cleaved during maturation. Whilst the signal peptide and transmembrane regions of the two proteins are very similar, the major hydrophilic domains are highly divergent in both size and sequence. The Amps from the two phytoplasmas are also different in structure and sequence from the immunodominant membrane proteins of three other phytoplasmas whose genes have been cloned previously.
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Immunological response mounted by Aboriginal Australians living in the Northern Territory of Australia against Streptococcus pyogenes serum opacity factor
The GenBank accession numbers for the sequences reported in this paper are AF367011 (sof VT3.2), AF367012 (sof VT3.1), AF367013 (sof VT2.2), AF367014 (sof VT21), AF367015 (sof VT37.1) and AF367016 (sof 13).
Streptococcus pyogenes (Group A streptococcus) interacts with host fibronectin via a number of distinct surface components. The streptococcal serum opacity factor (SOF) is a cell-surface protein of S. pyogenes which causes opalescence of human serum and mediates bacterial binding to fibronectin. In this study, hexahistidyl-tagged fusion proteins encompassing full-length SOF, and domains of SOF encompassing opacity factor activity and fibronectin-binding regions, were used in the characterization of the Aboriginal immune response to SOF. Anti-SOF serum IgG responses were found to be significantly higher (P<0·0001) in Aboriginal adults and children when compared to a non-Aboriginal adult group. The Aboriginal immune response against the fibronectin-binding region of SOF was significantly reduced when compared to the response against the whole SOF protein and N-terminal domains examined in this study (P<0·001). This pattern of immune response was also observed in rabbits immunized with recombinant SOF. Comparison of the deduced amino acid sequence of SOF from a number of common Australian isolates with other SOF sequences revealed that the N-terminus of SOF exhibits sequence similarity values ranging from 42·9% to 96·5%. The C-terminus containing the fibronectin-binding domain and membrane-spanning regions was more highly conserved, exhibiting sequence similarity values ranging from 84·6% to 100% within the fibronectin-binding repeats. These data suggest that the immune response against SOF is directed toward the variable N-terminus of the SOF protein. Phylogenetic analysis indicated that the sof genes of S. pyogenes do not exhibit geographical variation.
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Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis
More LessNitrogen fixation by diazotrophic bacteria is a significant source of new nitrogen in salt marsh ecosystems. Recent studies have characterized the physiological and phylogenetic diversity of oxygen-utilizing diazotrophs isolated from the rhizoplanes of spatially separated intertidal macrophyte habitats. However, there is a paucity of information regarding the traits encoded by and the diversity of plasmids occurring in this key ecological functional group. Five-hundred and twenty-one isolates cultivated from the rhizoplanes of Juncus roemarianus, Spartina patens and different growth forms (short-form and tall-form) of Spartina alterniflora were screened for the presence of plasmids. One-hundred and thirty-four diazotrophs carrying plasmids that ranged in size from 2 to >100 kbp were identified. The majority of the marine bacteria contained one plasmid. Diazotrophs from the short-form S. alterniflora rhizoplane contained significantly fewer plasmids relative to isolates from tall-form S. alterniflora, J. roemarianus and S. patens. Although some plasmids exhibited homology to a nifH gene probe, the majority of the plasmids were classified as cryptic. Two oligonucleotide primers were developed to facilitate genotypic typing of the endogenously isolated marine plasmids by the randomly amplified polymorphic DNA (RAPD)-PCR technique. These primers proved to be more effective than 21 commercially available primers tested to generate RAPD-PCR patterns. Analysis of the RAPD-PCR patterns indicated as many as 71 different plasmid genotypes occurring in diazotroph bacterial assemblages within and between the four different salt marsh grass rhizoplane habitats investigated in this study.
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Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans
The GenBank accession number for the RAS2 sequence of C. neoformans H99 is AF294349.
The Ras1 signal transduction pathway controls the ability of the pathogenic fungus Cryptococcus neoformans to grow at high temperatures and to mate. A second RAS gene was identified in this organism. RAS2 is expressed at a very low level compared to RAS1, and a ras2 mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The ras2 mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a ras1 ras2 double mutant strain is viable, mutation of both RAS genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the RAS2 gene completely suppressed the ras1 mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the ras1 mutant demonstrated actin polarity defects that were also partially suppressed by RAS2 overexpression. These studies indicate that the C. neoformans Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 °C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.
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Multiple origins of hybrid strains of Cryptococcus neoformans with serotype AD
More LessCryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial mAbs to capsular epitopes, strains of C. neoformans manifest five distinct serotypes – A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are thought to be haploid. In this study the origins and evolution of strains of serotype AD were investigated. A portion (537 bp) of the laccase gene was cloned and sequenced from 14 strains of serotype AD. Each strain contained two different alleles and sequences for both alleles were obtained. These sequences were compared to those from serotypes A, B, C and D. This analysis indicated that each of the 14 serotype AD strains contained two phylogenetically distinct haplotypes: one haplotype was highly similar to the serotype A group and the other to the serotype D group. To explain the origins of these serotype AD strains, genealogical analysis is consistent with at least three recent and independent hybridization events. The results demonstrate that the evolution of C. neoformans is continuing and dynamic.
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Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy
More LessAntisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 °C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.
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Evidence for degradation of 2-chlorophenol by enrichment cultures under denitrifying conditions
More LessAlthough chlorophenol (CP) degradation has been studied, no bacterium responsible for degradation of CP under denitrifying conditions has been isolated. Moreover, little substantial evidence for anaerobic degradation of CPs coupled with denitrification is available even for mixed cultures. Degradation of CP [2-CP, 3-CP, 4-CP, 2,4-dichlorophenol (DCP) or 2,6-DCP] under denitrifying conditions was examined in anaerobic batch culture inoculated with activated sludge. Although 3-CP, 4-CP, 2,4-DCP and 2,6-DCP were not stably degraded, 2-CP was degraded and its degradation capability was sustained in a subculture. However, the rate of 2-CP degradation was not significantly enhanced by subculturing. In 2-CP-degrading cultures, nitrate was consumed stoichiometrically and concomitantly during 2-CP degradation, and a dechlorination intermediate was not detected, suggesting that 2-CP degradation was coupled with nitrate reduction. A 2-CP-degrading enrichment culture degraded 2-CP in the presence of nitrate, but did not in the absence of nitrate or the presence of sulfate. This suggests that the enrichment culture strictly requires nitrate for degradation of 2-CP. The apparent specific growth rate of the 2-CP degrading species was 0·0139 d−1. Thus the apparent doubling time of the 2-CP-degrading population in the enrichment culture was greater than 50 d, which may explain difficulty in enrichment and isolation of micro-organisms responsible for CP degradation under denitrifying conditions.
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A broad-host-range vector of incompatibility group Q can work as a plasmid vector in Neisseria meningitidis: a new genetical tool
More LessPlasmid pHT128, a derivative of the broad-host-range IncQ vector pGSS33, was successfully introduced into Neisseria meningitidis. Under optimal conditions, pHT128 was transferred from Escherichia coli to N. meningitidis by triparental conjugation at a frequency of 10−5–10−6. The copy number of pHT128 in N. meningitidis was almost the same as in E. coli, in which the copy number of IncQ plasmids per chromosome is estimated to be 10. pHT128 was maintained as an episome in N. meningitidis in the presence of chloramphenicol, a marker of the plasmid. It was also shown that an opc or pilE1 gene cloned on pHT128 could be expressed in N. meningitidis under control of the tac promoter and could complement a mutation of opc or pilE1, respectively. In addition, the conjugational introduction of pHT128 into N. meningitidis was demonstrated to be independent of natural transformation competence. All the results indicate that pHT128 is a useful vector for N. meningitidis as a new genetical tool.
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Clostridium perfringens α-toxin induces rabbit neutrophil adhesion
More LessClostridium perfringens α-toxin, which is one of the main agents involved in the development of gas gangrene, stimulates production in neutrophils. Exposure of rabbit neutrophils to the α-toxin induced firm adhesion of the cells to fibrinogen and fibronectin. Incubation of rabbit neutrophils and neutrophil lysates with α-toxin led to the production of diacylglycerol (DG) and L-α-phosphatidic acid (PA), respectively. The toxin-induced DG and PA formation preceded the toxin-induced adhesion of the neutrophils to fibrinogen and fibronectin, and the production of . Pertussis toxin inhibited the α-toxin-induced formation of PA, the adhesion of the neutrophils to fibrinogen and production. GTPγS stimulated the events induced by the α-toxin, whereas GDPβS inhibited them. The α-toxin stimulated phosphorylation of a protein with a molecular mass of about 40 kDa. In addition, treatment of the cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12,13-dibutyrate (PDBu) stimulated cell adhesion, production of and phosphorylation of the 40 kDa protein, but had no effect on the formation of PA. The events induced by the presence of OAG and PDBu were not inhibited by pertussis toxin. Protein kinase C inhibitors, H-7, staurosporine and chelerythrine, blocked α-toxin-induced adhesion, production of and phosphorylation of the 40 kDa protein. These observations suggested that α-toxin-stimulated adhesion to the matrix and production were due to the formation of DG, through activation of phospholipid metabolism by a pertussis-toxin-sensitive GTP-binding protein, followed by activation of protein kinase C by DG.
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A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose
More LessThe GenBank accession number for the sequence determined in this work is AJ275975.
The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley β-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl β-D-cellotetraoside, blockage of the hydrolysis of NaBH4-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.
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Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set
More LessThe design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.
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Quantitative speciation of sulfur in bacterial sulfur globules: X-ray absorption spectroscopy reveals at least three different species of sulfur
X-ray absorption near edge structure (XANES) spectroscopy at the sulfur K-edge was applied to probe the speciation of sulfur of metabolically different sulfur-accumulating bacteria in situ. Fitting the spectra using a least-square fitting routine XANES reveals at least three different forms of sulfur in bacterial sulfur globules. Cyclooctasulfur dominates in the sulfur globules of Beggiatoa alba and the very recently described giant bacterium Thiomargarita namibiensis. A second type of sulfur globules is present in Acidithiobacillus ferrooxidans: here the sulfur occurs as polythionates. In contrast, in purple and green sulfur bacteria the sulfur mainly consists of sulfur chains, irrespective of whether it is accumulated in globules inside or outside the cells. These results indicate that the speciation of sulfur in the sulfur globules reflects the different ecological and physiological properties of different metabolic groups of bacteria.
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Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp.
More LessA phosphatase-overproducing Citrobacter sp. (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen. Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation. However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition. Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface.
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The metabolism of 2-methyladenosine in Mycobacterium smegmatis
2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.
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Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954
The GenBank accession number for the sequence reported in this paper is AF373598.
A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K m 3 μM) and activity with this substrate was inhibited by the presence of dicoumarol (K i 1·0 μM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K m 40 μM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21·5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K m for CB 1954 was lower than that of NTR (617 μM compared to 862 μM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).
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Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage
More LessDuring an infection of a higher eukaryote, dormant spores of a Bacillus species have been previously shown to be present in cells that can generate the toxic agent peroxynitrite (PON). Dormant spores of Bacillus subtilis were much more resistant to killing by PON than were growing cells, and spore-coat alteration or removal greatly decreased PON resistance. Spores were not killed by PON through DNA damage and lost no dipicolinic acid (DPA) during PON treatment. However, PON-killed spores lost DPA during subsequent heat treatments that caused much less DPA release from untreated spores. Although dead, the PON-killed spores germinated and initiated metabolism but never went through outgrowth; the great majority of germinated PON-killed spores also took up propidium iodide, indicating that they had suffered significant membrane damage and were dead. Together these data suggest that spore killing by PON is through some type of damage to the spore’s inner membrane.
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Establishment of a functional symbiosis between the cyanobacterium Nostoc punctiforme and the bryophyte Anthoceros punctatus requires genes involved in nitrogen control and initiation of heterocyst differentiation
More LessThe GenBank accession numbers for the coding regions of ntcA, hetR and hetF are AY038370, AF318069 and AF288130, respectively.
Three mutant strains (ntcA, hetR, hetF) of the cyanobacterium Nostoc punctiforme unable to differentiate heterocysts were characterized and examined for their ability to form a symbiotic association with the bryophyte Anthoceros punctatus. Previously unknown characteristics of the N. punctiforme hetR mutant include differentiation of chilling-resistant akinetes, while vegetative cells of the ntcA mutant randomly lysed, yielding short filaments, following ammonium deprivation. Strains with mutations in hetF and hetR infected A. punctatus with similar frequency to that of wild-type N. punctiforme but did not support growth of the plant partner. These results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association. They also indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocyst differentiation and nitrogenase expression. A strain with a mutation in the global nitrogen regulator ntcA did not infect A. punctatus despite its ability to differentiate hormogonia at a low frequency. When complemented with one or more copies of ntcA, the mutant strain infected A. punctatus at a similar frequency as the wild-type and supported growth of the plant partner in the absence of combined nitrogen. These results established a connection between the presence of a functional copy of ntcA and the magnitude of hormogonium differentiation, and the behaviour of the formed hormogonia.
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NAD(P)H regeneration is the key for heterolactic fermentation of hexoses in Oenococcus oeni
More LessOenococcus oeni (formerly Leuconostoc oenos) can perform malolactic fermentation, converting L-malate to L-lactate and carbon dioxide, in wines. The energy and redox potential required to support the growth of the micro-organism are supplied mainly by the consumption of carbohydrates via the heterolactic pathway. In the first steps of hexose metabolism two molecules of NAD(P)+ are consumed, which must be regenerated in later reactions. The aim of this work was to test if aerobic growth of O. oeni promotes higher cell yields than anaerobic conditions, as has been shown for other lactic acid bacteria. O. oeni M42 was found to grow poorly under aerobic conditions with glucose as the only carbohydrate in the medium. It was demonstrated that O2 inactivates the enzymes of the ethanol-forming pathway, one of the two pathways which reoxidizes NAD(P)+ cofactors in the heterolactic catabolism of glucose. These results suggest that the regeneration of cofactors is the limiting factor for the aerobic consumption of glucose. When external electron acceptors, such as fructose or pyruvate, were added to glucose-containing culture medium the growth of O. oeni was stimulated slightly; fructose was converted to mannitol, oxidizing two molecules of NAD(P)H, and pyruvate was transformed to lactate, enabling the regeneration of NAD+. The addition of cysteine seemed to suppress the inactivation of the ethanol-forming pathway enzymes by O2, enabling glucose consumption in aerobic conditions to reach similar rates to those found in anaerobic conditions.
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Methanol and acriflavine resistance in Dictyostelium are caused by loss of catalase
The GenBank accession number for the sequence reported in this paper is AF090443.
Various chemicals with harmful effects are not themselves toxic, but are metabolized in vivo to produce toxic products. One example is methanol in Dictyostelium, which is lethal to cells containing the acrA gene, but relatively harmless to acrA mutants. This makes methanol resistance one of the tightest genetic selections in Dictyostelium. Loss of acrA also confers cross-resistance to unrelated compounds such as acriflavine and thiabendazole. We have used insertional mutagenesis to demonstrate that the acrA locus encodes the peroxisomal catalase A enzyme. Disruption of the catA gene results in parallel resistance to acriflavine. Molecular and biochemical studies of several previously characterized methanol-resistant strains reveal that each lacks catalase activity. One allele, acrA2, contains a 13 bp deletion which introduces a frameshift in the middle of the gene. The involvement of catalase in methanol resistance in Dictyostelium compares with its role in methanol metabolism in yeast and rodents. However, this is the first study to show that catalase is required for the toxicity of acriflavine. Our results imply that acriflavine and thiabendazole are precursors which must be oxidized to generate biologically active species. The catA/acrA gene is also a potentially invaluable negative selectable marker for Dictyostelium molecular genetics.
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