- Volume 145, Issue 7, 1999
Volume 145, Issue 7, 1999
- Microbiology Comment
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- Antigens And Immunity
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Both CD4+ and CD8+ lymphocytes reduce the severity of tissue lesions in murine systemic candidiasis, and CD4+ cells also demonstrate strain-specific immunopathological effects
More LessThe role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4+ and CD8+ cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4+ lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8+ cells. There were no major differences between days 4 and 8 after infection in cDNA cytokine profiles of CD4+ lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4+ and CD8+ lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4+ lymphocyte effector function is influenced by the genetic background of the mouse.
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- Biochemistry
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The diacetamidodideoxyuronic-acid-containing glycan chain of Bacillus stearothermophilus NRS 2004/3a represents the secondary cell-wall polymer of wild-type B. stearothermophilus strains
The diacetamidodideoxymannuronic-acid-containing glycan of Bacillus stearothermophilus NRS 2004/3a with the repeating unit structure [→ 4)-β-d-ManpA2,3(NAc)2-(1 → 6)-α-d-Glcp-(1 → 4)-β-d-ManpA2,3(NAc)2-(1 → 3)-α-d-GlcpNAc-(1 →], was examined to identify its linkage to the bacterial cell wall. In a previous paper it was suggested that this glycan is covalently linked to the surface layer (S-layer) glycoprotein of that organism. By improved chromatographic techniques (gel permeation over Sephacryl S-1000 SF; C4 reversed-phase HPLC) the diacetamidodideoxyuronic-acid-containing material was completely separated from the S-layer glycoprotein. This implicates only low, if any, specific affinity between these cell-wall components. To obtain sufficient amounts for the chemical characterization of its linkage region, the identical diacetamidodideoxyuronic-acid-containing material was isolated from sonicated cells of that organism by a purification procedure different to that for preparation of S-layers. This method allowed collection of the intact molecule including its linkage region. From the combined results of the chemical characterization and 600 MHz NMR spectroscopy it is proposed that the diacetamidodideoxyuronic-acid-containing glycan chain, consisting of approximately six tetrasaccharide repeating units, is directly linked via a pyrophosphate bridge to carbon 6 of muramic acid residues of the peptidoglycan sacculus. About 20–25% of the muramic acid residues are substituted with these polysaccharide chains. Thus, the diacetamidodideoxyuronic-acid-containing glycan represents a secondary cell-wall polymer of B. stearothermophilus NRS 2004/3a.
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Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152
More LessIn cultures of the unicellular cyanobacterium Gloeothece sp. ATCC 27152 growing under alternating 12 h light and 12 h darkness, nitrogenase activity appears as cultures enter the dark phase. Synthesis of both component proteins of nitrogenase commences immediately prior to the appearance of activity and continues until about 8 h into the period of darkness. The two components (Fe-protein and MoFe-protein) are synthesized in a molar ratio of about 3:1. Degradation of the nitrogenase proteins starts as early as 4 h into the dark period and increases markedly as cultures enter the light phase. As a result, both nitrogenase proteins are completely absent from cultures during most of the light phase. In contrast, all of the other proteins investigated appeared to be present throughout the cycle of alternating light and darkness. Degradation of nitrogenase depends upon protein synthesis during the last 6 h of darkness and is prevented by addition of protease inhibitors. Two proteins, of M r 47000 and 29000, are specifically synthesized during this period and it is possible that they have a role in nitrogenase degradation. Proteolytic activity of extracts of Gloeothece, measured as the ability to degrade azocasein, increased markedly during the early part of the light period, but this increase did not depend on protein synthesis. This activity does not therefore correspond to that specifically involved in nitrogenase catabolism, though it may act on initial breakdown products generated by a nitrogenase-specific degradative system. A phycobiliprotein appears to act as a temporary store of the degradation products of nitrogenase.
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- Bioenergetics And Transport
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An unusual cytochrome o′-type cytochrome c oxidase in a Bacillus cereus cytochrome a 3 mutant has a very high affinity for oxygen
Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa 3 or caa 3 . The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied. Respiratory oxidase activities and the levels of cytochromes b and c increased 2·6- to 4·2-fold on transition from exponential growth, in either of two media, to sporulation stage III, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethyl-p-phenylenediamine (TMPD) oxidase activity, consistent with the TMPD− phenotype of strain PYM1. Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron mediator, suggesting that the terminal oxidase receives electrons from a cytochrome c. Carbon monoxide (CO) difference spectra of membranes were obtained using various reductants (ascorbate±TMPD, NADH, dithionite) and revealed a haemoprotein resembling cytochrome o′. The CO complex of this cytochrome was photodissociable: the photodissociation spectrum (photolysed minus CO-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the α/β region of the spectrum, consistent with the presence of a cytochrome o′-like pigment. CO recombination occurred at −85 to −95°C. No other haemoproteins showing photoreversible CO binding under these conditions were detected. Evidence that this pigment was the oxidase responsible for substrate oxidation was obtained by photodissociating the CO complex at subzero temperatures in the presence of oxygen; this resulted in faster ligand recombination, attributed to oxygen binding, and extensive oxidation of cytochromes c and b. The oxygen affinity of the oxidase was determined by using the deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissociated oxygen concentration. A single oxidase was revealed with a K m for oxygen of about 8 nM; this is one of the highest affinities yet reported for a terminal oxidase.
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- Development And Structure
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FliL is a membrane-associated component of the flagellar basal body of Salmonella
More LessFliL is one of the least understood proteins in the flagellar systems of Salmonella and Escherichia coli. There is no apparent mutant phenotype associated with it, even when virtually the entire coding sequence has been eliminated. In this study it has been shown that FliL is a cytoplasmic membrane protein associated with the basal body. Although it has a sequence that conforms to the consensus cleavage site for lipoproteins, FliL does not undergo cleavage or modification under physiological conditions.
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- Environmental Microbiology
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Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes
More LessBacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G+C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C23O). C23O specific activities were very diverse, ranging from 0·1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C23O, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.
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- Genetics And Molecular Biology
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Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis
More LessDetailed functional analysis revealed the modular organization of pGI2, a 9672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp transposon Tn4430. Whereas the pGI2 leading-strand replicon was identified through deletion experiments, sequence comparisons indicated the presence of an sso t-like single-strand origin commonly found among Bacillus plasmids. Southern hybridization confirmed the existence of ssDNA intermediates, but only in the case of plasmid derivatives lacking the sso t site. Moreover, the pGI2 replication protein Rep displayed significant similarity with that of pTX14-3, a 7·6 kb plasmid from B. thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolling-circle replicating (RCR) plasmids. In addition, both plasmids share a conserved 320 bp region downstream of their rep genes which, in the case of pGI2, appeared indispensable for replication. This region is therefore likely to correspond to, or to be part of, the actual double-strand origin of both plasmids. Another interesting feature of pGI2 is the presence of a mobilization (Mob) protein, as demonstrated by its ability to be mobilized by the conjugative plasmid pAW63 from B. thuringiensis serovar kurstaki HD73. The same transfer system was also used to unambiguously demonstrate similar properties of the related Mob-like protein from pTX14-3. A closer analysis of this family of related Mob proteins suggested a subdomanial organization among its members. Finally, the 270 residue pGI2 ORF2 was shown to be related to ORF43 of pMRC01, a 60 kb conjugative plasmid from Lactococcus lactis subsp. lactis. Although no function has been assigned to the putative ORF43 protein, it is located downstream of a bacteriocin operon, next to an IS946 element. pGI2 appears thus far as an assemblage of functional modules with no obvious metabolic function, presumably acting as a reservoir of carrier (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for its bacterial host.
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Erwinia carotovora has two KdgR-like proteins belonging to the IcIR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis
More LessA novel Erwinia carotovora subsp. carotovora mutant designated RexZ, (regulator of exoenzymes) showed reduced production of the degradative exoenzymes. The rexZ gene product shows similarity to the KdgR regulatory protein from Erwinia chrysanthemi, described as the major repressor of the pectin catabolism pathway genes in the latter species. In vitro DNA—protein interaction experiments demonstrated that the synthesis of the RexZ protein is controlled by the cAMP—CRP (cAMP—receptor protein) complex. Western blot analysis also revealed the presence of a second KdgR homologue (distinct from RexZ) which, like RexZ, was present in all species of the genus Erwinia tested. The corresponding KdgR proteins from both E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica share a high level of sequence identity with the KdgR homologues from E. chrysanthemi and Escherichia coli. Although the E. carotovora subsp. carotovora rexZ regulatory region displayed specific interactions with both the purified E. chrysanthemi KdgR repressor and the partially purified E. carotovora subsp. carotovora KdgR, in vivo quantification revealed that the cellular level of RexZ protein was unaffected by the presence of pectic compounds. This study shows that the complex regulatory network governing virulence in the erwinias involves two totally distinct, but highly conserved, members of the IcIR class of DNA binding proteins: RexZ and KdgR.
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Ribonucleotide reductase (RNR) of Corynebacterium glutamicum ATCC 13032–genetic characterization of a second class IV enzyme
More LessRibonucleotide reductases (RNRs) encoded by nrd (nucleotide reduction) genes are unique enzymes providing the DNA precursors in all living organisms and several viruses. The designation of four classes of RNRs reflects their use of diverse metallo-cofactors. Using oligonucleotide primers derived from conserved domains of the primary structure of known NrdA and NrdE proteins, an internal 938 bp fragment of the nrdE gene was amplified from genomic DNA of Corynebacterium glutamicum. With this PCR product a 4·36 kb fragment was identified and cloned containing the nrdHIE genes of C. glutamicum. A probe derived from nrdF2 of Mycobacterium tuberculosis allowed the cloning and sequencing of the nrdF gene located 3·1 kb further downstream, encoding the small subunit of the C. glutamicum RNR. Conjugative introduction of nrdE from C. glutamicum complemented thermosensitive mutants of Corynebacterium ammoniagenes which had a defective catalytic subunit of the Mn-RNR. The authors provide arguments for allocation of the C. glutamicum NrdEF proteins to class IV in the RNR classification scheme of Stubbe & van der Donk (1995) [Chem Biol 2, 793-801].
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The nuclear rDNA intergenic spacer of the ectomycorrhizal basidiomycete Laccaria bicolor: structural analysis and allelic polymorphism
More LessThe nuclear rDNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Laccaria bicolor was amplified and sequenced to identify the source of its intraspecific polymorphism. The IGS was amplified by PCR in several L. bicolor strains and shown to exhibit multiple bands and length polymorphism. The IGS loci were shown to segregate in a 1:1 ratio within haploid progenies in three dikaryotic strains, suggesting that divergent IGS haplotypes were present in the two nuclei of these strains. The two haplotypes of L. bicolor S238N were sequenced: the β-haplotype was 4160 bp in length, whereas the size of the α-haplotype was estimated to be about 4700 bp. These represent the largest published fungal IGS sequences to date. These sequences can be subdivided into three main regions, IGS1, 5S rDNA and IGS2. The IGS sequences are AT-rich and contain numerous occurrences of three types of subrepeats (e.g. T2AC3). The length polymorphism, observed between the IGS sequence of the α- and β-haplotypes, results from the insertion of various numbers of a 71 bp subrepeat, called B, occurring in IGS2. This variation in subrepeat number suggests that the two haplotypes resulted from unequal cross-overs. The L. bicolor IGS was aligned with IGS sequences of two other Tricholomataceae (i.e. Tricholoma matsutake and Collybia fusipes). No sequence similarity was observed between these IGSs, but homologous subrepeats were found in L. bicolor and T. matsutake. Analysis of IGS length polymorphism is therefore an efficient tool for investigating genetic relationships between genets and within progenies in natural fungal populations.
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The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis
The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61 % identity in the C-terminal half of the proteins and that region of C. albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis.
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Loss of heterozygosity, by mitotic gene conversion and crossing over, causes strain-specific adenine mutants in constitutive diploid Candida albicans
More LessMolecular evidence is provided in this paper to elucidate a long-standing intriguing phenomenon in fungal genetics: that many natural isolates of the constitutive diploid organism Candida albicans yield strain-specific, recessive mutants at a reproducible frequency that is as high as a few percent of the surviving cells after exposure to UV irradiation or other mutagens. Southern hybridization analysis and DNA sequence data indicated that C. albicans CA12, a clinical isolate, is heterozygous for the ADE2 gene, carrying one functional and one null allele. Sequence analysis of the null allele revealed the presence of a 1·3 kb deletion, which locates between two AATC repeats and spans the promoter and coding regions of the gene. The adenine auxotrophic mutants, which were readily isolated after UV irradiation of C. albicans CA12, were proved to be the segregants of mitotic recombination as they remained as diploid, not hemizygous or haploid, cells and were homozygous for ade2. Analysis of reciprocal products of the mitotic recombination detected that the process of loss of heterozygosity was mediated by mitotic crossing over (reciprocal exchange of genetic information) as well as gene conversion (non-reciprocal exchange of genetic information).
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The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases
More LessThe temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3-α-l-rhamnosidase (endorhamnosidase) activity which results in cleavage of the lipopolysaccharide O-antigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene 9 (encoding P22 TSP) DNA probe hybridized with restriction fragment Pst1-7 of Sf6. DNA sequencing and analysis of Pstl-7 and the adjacent Psrl-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP N-terminal head-binding domain. High conservation of key residues was suggestive of similar secondary and tertiary N-terminal protein structure and a similar function of the Sf6 TSP in this region. In addition, an amino acid sequence motif (DFGX3DGX6AX3A) was identified between residues 164 and 184 which was also found to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by l-[35S]methionine labelling and SDS-PAGE). Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole Sh. flexneri Y cells demonstrated that O-antigen hydrolysis activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific and preferential activity for long-chain Sh. flexneri serotype X or Y O-antigen, cleavage of which resulted in the release of oligosaccharide fragments, consistent with octasaccharides in size, as detected by fluorophore-assisted carbohydrate electrophoresis (FACE).
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DNA structure and novel amino and carboxyl termini of the Chlamydia σ70 analogue modulate promoter recognition
More LessGenes from the eubacterium Chlamydia typically do not share promoter consensus sequences with those of Escherichia coli and are not expressed when cloned in E. coli; nevertheless, the major σ-subunit identified from Chlamydia trachomatis has nearly identical amino acid sequence to E. coli σ70 in regions that contact DNA. Following expression of the chlamydial σ-subunit gene in E. coli, expression was specifically initiated from chlamydial promoter regions. Selective recognition of chlamydial promoters by holoenzyme was dependent upon the structure of the promoter DNA coupled with novel amino-and carboxyl-terminal extensions of the chlamydial σ-subunit.
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Random mutagenesis of the pomA gene encoding a putative channel component of the Na+-driven polar flagellar motor of Vibrio alginolyticus
More LessPomA and PomB are integral membrane proteins and are essential for the rotation of the Na+-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H+-driven motor, they are thought to form the Na+-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T186I is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.
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- Genomics
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Genomic structure of phage B40-8 of Bacteroides fragilis
More LessVery few data are available on the molecular biology of Bacteroides fragilis bacteriophages, which have been considered in several studies as indicators of faecal contamination. Phage B40-8, initially isolated from an urban sewage sample using a strain of B. fragilis (HSP40) isolated from a clinical specimen, was chosen in this study as a prototype for morphological and molecular studies. Like most of the phages infective for B. fragilis, B40-8 belongs to the Siphoviridae family. Its genome has been found to be a double-stranded DNA molecule, of approximately 51·7 kb, containing a rather low percentage (38·9 mol%) of G+C. The ends of the molecule appeared not to be cohesive but permuted, with a terminal redundancy of 7·3%. A genomic map was constructed. Three major proteins (MP) out of 15 peptides in the SDS-PAGE profile were selected for N-terminal sequencing. From these data, degenerate probes were designed to locate the ORFs in the genomic map. Immunodetection by electron microscopy revealed that MP1 and MP3 were structural proteins of the phage head and that MP2 was a constituent of the tail. A genomic library of the phage was prepared, and a clone including the MP2 ORF was identified and sequenced.
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A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex
It has previously been shown that the PAN promoter from Mycobacterium paratuberculosis can be used as a DNA probe to identify an RFLP between wild-type Mycobacterium bovis and the vaccine strain Mycobacterium bovis BCG. To investigate the genetic basis of this phenomenon, DNA fragments from a New Zealand M. bovis cattle strain and M. bovis BCG Pasteur, containing the PAN-binding region, were isolated from gene libraries, sequenced and characterized. Sequence analysis and comparison with database sequences showed that the PAN region in M. bovis, M. bovis BCG and Mycobacterium tuberculosis is identical and shares 70% similarity to the PAN sequence from M. paratuberculosis. The Shine-Dalgarno sequence and the −10 and −35 promoter regions are conserved between the different species. Analysis of the flanking sequences of the PAN region revealed that less than 1 kb downstream of PAN is a 2405 bp fragment that is present in M. bovis BCG but absent in the M. bovis wild-type strain. The distribution of the 2405 bp fragment in members of the M. tuberculosis complex was investigated and found to be present in 70 out of 70 M. tuberculosis strains, and 7 out of 7 M. bovis BCG daughter strains, 2 out of 2 Mycobacterium africanum strains, 2 out of 2 Mycobacterium microti strains and 7 out of 25 M. bovis strains. This is the first report of a genetic region of M. bovis BCG that is not universally present in M. bovis strains. The fragment does not appear to be present in any mycobacterial species outside the M. tuberculosis complex. It does not possess any characteristics of known transposable elements and the flanking sequences do not have any obvious features to suggest a deletion mechanism. The genetic location of this region is close to the 3′ end of the RD1 region of M. bovis and M. tuberculosis. The polymorphic nature of this locus in M. bovis will provide an additional genetic marker for strain differentiation.
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- Pathogenicity And Medical Microbiology
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Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae
More LessMembers of the taxonomically diverse Burkholderia cepacia complex have become a major health risk for patients with cystic fibrosis (CF). Although patient-to-patient transmission of B. cepacia strains has been well-documented, very little is known about possible vehicles of transmission and reservoirs for these micro-organisms. In this work, it is shown that strains of the B. cepacia complex can survive within different isolates of the genus Acanthamoeba. Trophozoites containing bacteria developed profuse cytoplasmic vacuolization. Vacuolization was not detected in trophozoites infected with live Escherichia coli or heat-killed B. cepacia, or by incubation of trophozoites with filter-sterilized culture supernatants, indicating that metabolically active intracellular bacteria are required for the formation of vacuoles. Experiments with two different B. cepacia strains and two different Acanthamoeba isolates revealed that bacteria display a low level of intracellular replication approximately 72–96 h following infection. In contrast, extracellular bacteria multiplied efficiently on by-products released by amoebae. The findings suggest that amoebae may be a reservoir for B. cepacia and possibly a vehicle for transmission of this opportunistic pathogen among CF patients.
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In vitro survival and germination of Candida albicans in the presence of nitrogen compounds
The in vitro effect of nitric oxide (NO) and nitrite on blastoconidia and hyphae of Candida albicans was studied. Sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) were used as NO donors. Both minimal and complex media at two pH values, 7·0 and 4·5, were used for the assays. Blastoconidia were more susceptible than hyphae to NO. The NO effect on blastoconidia was greater at acidic pH. Nitrite affected the viability of blastoconidia in complex medium. The percentage germination and the relative rate of elongation of hyphae were both enhanced when NO was present in acidic conditions.
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Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids
More LessThe impact of various growth conditions on the expression of toxins and other proteins by Clostridium difficile VPI 10463 was studied. During non-starved conditions, the rate of toxin synthesis paralleled that of total protein during both exponential growth and stationary phase, and in both defined and complex media. Biotin limitation reduced growth rate and bulk protein synthesis, whereas toxin expression continued, leading to a 50- to 200-fold increase in intracellular toxin levels. Concomitantly, several 22 kDa proteins were up-regulated as revealed by two-dimensional PAGE analysis. The toxin yield was 30-fold higher in peptone yeast extract (PY) than in PY containing glucose (PYG). By contrast glucose limitation reduced toxin yields by 20- to 100-fold in defined media. By elevating the buffering capacity and bicarbonate concentration, toxin yields were increased by 10-fold in PY and PYG. The high toxin production by C. difficile during growth in PY was lowered 100-fold by adding a blend of nine amino acids and several 60-100 kDa proteins were concomitantly down-regulated. It was concluded that toxin expression in C. difficile VPI 10463 was not affected by growth rate, growth phase, catabolite repression or the stringent response. Instead the co-expression of toxins and a few specific additional proteins appeared to be influenced by metabolic pathways involving CO2 assimilation, carboxylation reactions and metabolism of certain amino acids.
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Identification of IngA, the structural gene of longus type IV pilus of enterotoxigenic Escherichia coli
Human enterotoxigenic Escherichia coli (ETEC) produces a type IV pilus termed longus which is encoded on large plasmids in association with colonization factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHI DNA fragment hybridizing with an oligonucleotide probe designed from the amino-terminal amino acid sequence of the denatured 22 kDa structural longus pilin subunit was subcloned and sequenced. DNA sequencing analysis revealed an open reading frame, designated IngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. IngA is the first gene described of the longus gene cluster. Cloned IngA encoded and expressed a prepilin protein of 236 residues with a calculated mass of 25·17 kDa. The prepilin is apparently processed into a mature pilin of 206 residues with a calculated mass of 21·5 kDa. The predicted peptide sequence of IngA showed 78·8 and 37% identity to CFA/III pilin (CofA) of ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide sequence homology between IngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA contains two cysteine residues towards the carboxy-terminal region. Transmission electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFA/III detected antigenic differences between native and denatured epitopes of these pili. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different media. Our data indicate that longus and CFA/III are two distinct but yet highly related type IV pili of ETEC.
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- Physiology And Growth
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Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions
More LessIt has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role, in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.
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Survival and exopolysaccharide production in Sinorhizobium meliloti WSM419 are affected by calcium and low pH
More LessCells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown at pH 5·8 instead of pH 7·0. Increasing concentrations of calcium in the exposure medium significantly decreased the death rate of WSM419 cells under conditions of acid stress (pH 4·0). The effect of calcium on survival at pH 4·0 however, appears unconnected to exopolysaccharide (EPS), since a strain with a mutation in exoY (Rm0540) responded to calcium in the exposure medium in the same way as its wild-type parent (Rm2011). The concentration of calcium in the growth medium also affected subsequent survival at pH 4·0, and the effect varied with pH. In cells grown at pH 5·8, higher calcium concentrations also markedly increased the rate of synthesis of EPS; this was not seen in cells grown at pH 7·0. 1H NMR spectra for isolated EPS from WSM419 cultures grown at pH 5·8 and pH 7·0 showed that low pH markedly lowered the degree of substitution with acetyl and pyruvyl groups, but not the degree of substitution with succinyl groups; calcium concentration did not affect the pattern of substitution at either pH. For EPS to be involved in the effect of calcium concentration in the growth medium on survival would imply a deleterious effect of the EPS produced at low pH.
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Use of fluorescence ratio imaging for intracellular pH determination of individual bacterial cells in mixed cultures
More LessThe development of a rapid method for measuring intracellular pH (pHi) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R490/435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pHi of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pHex) values, an in vivo calibration curve was constructed in the pHi range 5·0–8·5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pHi almost independently of pHex (5·0–8·0), whereas the pHi of Lb. delbrueckii subsp. bulgaricus decreased with decreasing pHex. In pure and mixed cultures at pHex 5·0, the pHi values of Lb. delbrueckii subsp. bulgaricus and L. innocua were 6·1 ± 0·2 and 7·5 ± 0·2, respectively. This difference in pHi may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pHex.
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The effect of heavy metals and other environmental conditions on the anaerobic phosphate metabolism of Acinetobacter johnsonii
More LessA strain of Acinetobacter with potential for bioremediation of heavy metal-contaminated waters was isolated from a wastewater-treatment plant operating an enhanced biological phosphate removal process. NMR and extractive methods showed that polyphosphate accumulated aerobically was degraded under anaerobic conditions both in the presence and absence of cadmium or uranium (0·2–0·5 mM). NMR showed that free phosphate was formed at the expense of polyphosphate, and an extractive technique indicated that this reaction could be stimulated by the presence of UO2 2+ under these conditions. Energy-dispersive X-ray microanalysis demonstrated that only cadmium could enter the cells, and co-localized with intra-cellular granules containing phosphate and other divalent metals. The effects of other environmental parameters on the anaerobic phosphate metabolism were also investigated. Between pH 5·5 and 8·0, phosphate release increased with increasing pH. Between 4°C and 37°C, phosphate release increased with increasing temperature. The presence of nitrate at concentrations of 10 mM and above inhibited anoxic phosphate release, but supplying tungstate in the growth medium prior to anoxic incubation reduced the production of active nitrate reductase and alleviated this effect.
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Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276
More LessRhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K m and V max values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 μM and 2·4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains.
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Evidence for the presence of the reductive pentose phosphate cycle in a filamentous anoxygenic photosynthetic bacterium, Oscillochloris trichoides strain DG-6
Studies on autotrophic CO2 fixation by the filamentous anoxygenic photosynthetic bacterium Oscillochloris trichoides strain DG-6 demonstrated that, unlike other green bacteria, this organism metabolized CO2 via the reductive pentose phosphate cycle. Both key enzymes of this cycle — ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase — were detected in cell extracts. The main product of ribulose 1,5-bisphosphate-dependent CO2 fixation was 3-phosphoglyceric acid. KCN, which is known to be a competitive inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, completely in hibited the CO2 assimilation by whole cells as well as by cell extracts of O. trichoides. The 13C/12C carbon isotope fractionation during photoautotrophic growth of O. trichoides was -19·7°/∞, which is close to that obtained for autotrophic organisms that use ribulose-1,5-bisphosphate carboxylase as the primary carboxylation enzyme. Cell extracts of O. trichoides contained all the enzymes of the tricarboxylic acid cycle except 2-oxoglutarate dehydrogenase. No activity of isocitrate lyase, a key enzyme of the glyoxylate shunt, was found in cell extracts of O. trichoides DG-6.
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- Systematics And Evolution
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Prototheca richardsi, a pathogen of anuran larvae, is related to a clade of protistan parasites near the animal-fungal divergence
More LessPrototheca richardsi is a protist of uncertain taxonomy which mediates growth inhibition in anuran larvae. Cells of P. richardsi were isolated from tadpole faeces and DNA was purified by Qiagen chromatography. Nuclear small-subunit (18S) rDNA (ssu-rDNA) was amplified by PCR using universal primers, cloned, and six clones (two from each of three separate isolates) were sequenced. All clones yielded an essentially identical sequence of 1802 nucleotides. In situ hybridization of fluorescent Prototheca-specific oligonucleotide probes, designed using the derived 18S rDNA sequence, confirmed that the sequence was indeed from P. richardsi cells and not from other components of tadpole faeces. The P. richardsi sequence was aligned with ssu-rDNA from a range of other eukaryotes, and phylogenetic analyses were carried out using several inference methods. P. richardsi consistently and stably grouped within a novel clade that contains rDNAs from an apparently heterodisperse group of parasitic micro-organisms assigned to the class Ichthyosporea. P. richardsi is evidently misplaced in the genus Prototheca, and the authors propose its inclusion in a new genus Anurofeca.
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DNA sequence heterogeneity in the three copies of the long 16S–23S rDNA spacer of Enterococcus faecalis isolates
More LessThe possibility of intragenic heterogeneity between copies of the long intergenic (16S–23S rDNA) spacer region (LISR) was investigated by specific amplification of this region from 21 Enterococcus faecalis isolates. Three copies of the LISR (rrnA, B and C) were demonstrated by hybridization of the LISR to genomic DNA cleaved with I-Ceul and Smal. When the LISR amplicon was digested with Tsp5091, two known nucleotide substitutions were detected, one 4 nt upstream from the 5′ end of the tRNAala gene (allele rrnB has the Tsp509l site and rrnA and C do not) and the other 22 nt downstream from the 3′ end of the tRNAala gene (rrnC has the Tsp509l site). Sequence differences at these sites were detected at the allelic level (alleles rrnA, B and C) and different combinations of these alleles were designated Tsp Types. Using densitometry to analyse bands from electrophoresis gels, the intra-isolate ratios of the separate alleles (rrnA:rrnB:rrnC) were determined in each Tsp Type: I (0:3:0), II (1:2:0), III (2:0:1), IV (3:0:0), V (2:1:0) and VI (1:1:1). Sequence variation between the three copies of the LISR was confirmed by the detection of at least five other intra-isolate nucleotide substitutions using heteroduplex analysis by conformation-sensitive gel electrophoresis (CSGE) that were not detected by Tsp509I cleavage. Perpendicular denaturing gradient gel electrophoresis was capable of resolving homoduplexes; six to seven out of a possible nine curves were obtained in some isolates. In the isolate where seven curves were obtained one or more further nucleotide substitutions, not detected by Tsp509l cleavage or CSGE, were detected. On the basis of LISR sequence heterogeneity, isolates were categorized into homogeneous (only one allele sequence present) and heterogeneous (two or three allele sequences present). The transition between homogeneous and heterogeneous LISRs may be useful in studying evolutionary mechanisms between E. faecalis isolates.
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16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep
The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen. Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens. All sequences fell into cluster IV of the Clostridia, while other species of ruminococci (eg. Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XlVa of the Clostridia. Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment. A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes. A combination of the Ruminococcus and F. succinogenes probes accounted for 4·0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5·2% of the total culturable count. These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences. Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained. These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.
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