- Volume 140, Issue 8, 1994
Volume 140, Issue 8, 1994
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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Lipopolysaccharide in cells infected by Chlamydia trachomatis
More LessIn view of the controversy concerning the expression of chlamydial lipopolysaccharide (LPS) in the eukaryotic host cell, the presence of this molecule was examined in three cell types which were experimentally infected with Chlamydia trachomatis serotype E. LPS was detected in the McCoy cell line, human endometrial epithelium and human amniotic epithelium with two monoclonal antibodies. The appearance and distribution of LPS at the host cell surface during the chlamydial developmental cycle and its transmission to neighbouring cells were examined by immunofluorescence microscopy after air drying of the host cells. LPS distribution was not uniform; it was first observed on regions of the cell surface in close proximity to the chlamydial endosome (inclusion). Soon after, the antigen was also detected at points of contact with neighbouring uninfected cells. Immunofluorescent plaques of host cells contaminated with LPS were thus formed in the vicinity of infected cells. These plaques increased in size over 2 d before becoming smaller as host cell lysis occurred. The major outer-membrane protein (MOMP) was not visualized on the host cell surface after air drying. No cell-surface LPS antigen was observed in live cells or those fixed in formaldehyde without air drying. Conventional methanol fixation and immunolocalization of LPS and MOMP in parallel infected cultures stained these antigens within inclusions in the expected fashion. Radio-immunoassays were used to quantify LPS in confluent McCoy cell monolayers during the chlamydial developmental cycle. Cell-surface-associated and inclusion-associated LPS, measured by direct binding of 125I-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined. Cell-surface-associated LPS is not directly accessible to antibodies in the hydrated cell. This apparent masking of the antigen may have a significant advantage for persistence of the parasite in vivo, since such host-cell-associated antigen is unlikely to be a target for immune attack.
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Immunoreactivity of the 60 kDa cysteine-rich proteins of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae expressed in Escherichia coli
More LessThe 60 kDa cysteine-rich proteins (CrPs) of Chlamydia are developmentally regulated outer envelope proteins synthesized late in the chlamydial growth cycle. These proteins, found only on the extracellular infectious elementary bodies, elicit major antibody responses in chlamydial infection. We have cloned and expressed in Escherichia coli the complete 60 kDa CrP genes from Chlamydia trachomatis, C. psittaci and C. pneumoniae. The recombinant products were expressed as either ‘native’ proteins or as fusions with the bacteriophage T7 gene 10 protein. Electron microscopy showed that recombinant proteins were produced as insoluble inclusions within the E. coli host cells. The recombinant 60 kDa CrPs were purified and used to raise high titre polyclonal antisera. In immunoblot analysis these antisera reacted with the 60 kDa CrPs from purified elementary bodies of all three chlamydial species in a genus-specific manner. Further molecular analysis allowed the genus-specific cross-reacting epitopes to be localized by using overlapping synthetic peptides covering the C. trachomatis 60 kDa CrP. Immunogold labelling experiments using purified infectious elementary bodies from the three chlamydial species indicated that the 60 kDa CrPs are not surface accessible to antibody binding.
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- Biochemistry
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Biochemical characterization of Bacillus thuringiensis cytolytic δ-endotoxins
More LessThe entomocidal δ-endotoxins CytA and CytB produced by Bacillus thuringiensis (Bt) subspecies israelensis and kyushuensis respectively showed a similar level of toxicity to mosquito larvae but were not toxic to the larvae of the lepidopteran Manduca sexta. CytA and CytB are also similar in sequence, predicted secondary structure and α-helical content, the only obvious difference being a C-terminal fifteen residue ‘tail’ on CytB. Investigations of the function, if any, of the CytB C-terminal ‘tail’ showed that this δ-endotoxin is highly expressed and forms inclusions in an acrystalliferous Bt mutant without the aid of the 20 kDa ‘helper’ protein from Bt subspecies israelensis which is essential for CytA inclusion formation. After proteinase K treatment, CytA and CytB were processed to virtually the same points in a sequence alignment and were equally haemolytic in vitro. However, the results suggested that unprocessed CytB differs from unprocessed CytA in that the former is not haemolytic.
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Lipids of extremely halophilic archaeobacteria from saline environments in India: A novel glycolipid in Natronobacterium strains
More LessSeveral strains of extremely halophilic archaeobacteria, both non-alkaliphilic and alkaliphilic, including Halobacterium, Haloferax and Natronobacterium species, were isolated from salt locales in India. The major phospholipids in these strains were the C20-C20-glycerol diether analogues of phosphatidylglycerolmethylphosphate (PGP-Me), phosphatidylglycerol (PG) and phosphatidic acid (PA). In addition, the Halobacterium strains possessed the characteristic glycolipids, sulfated triglycosyl and tetraglycosyl diethers (S-TGD-1 and S-TeGD, respectively) and the unsulfated triglycosyl diether (TGD-1); and the Haloferax strains had the characteristic sulfated and unsulfated diglycosyl glycerol diethers (S-DGD-1 and DGD-1, respectively). The PGP-Me, and PG components of the haloalkaliphiles each occurred as two molecular species with C20-C20- and C20-C25- (isopranoid) glycerol diether lipid cores. In contrast to previous reports of the absence of glycolipids in natronobacteria, the Natronobacterium strains from India were found to contain small amounts of a novel glycolipid identified as glucopyranosyl-1 → 6-glucopyranosyl-1 →1-glycerol diether (DGD-4). The lipid cores of DGD-4 also contained mainly unhydroxylated or hydroxylated C20-C20, C20-C25 and C25-C25 molecular species with unsaturated (isoprenoid) chains. Hydroxylated lipid cores have previously been identified only in some methanogenic archaeobacteria.
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The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi, a new deep-sea hyperthermophilic archaeobacterium
More LessThe catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi were studied in the GE5 strain isolated from a deep-sea hydrothermal vent located in the North-Fiji Basin in the SW Pacific Ocean. The enzyme from this hyperthermophilic archaeobacterium shows homotropic cooperative interactions between catalytic sites for the utilization of its two substrates, carbamoylphosphate and aspartate. The activity of this enzyme is subject to allosteric regulation. It is feed-back inhibited by the end-product cytidine triphosphate independently of temperature. In contrast, its sensitivity to the feed-back inhibitor uridine triphosphate and to the activator adenosine triphosphate disappears at high temperature. The unusual response of this aspartate transcarbamoylase to carbamoylphosphate analogues suggests a particular mode of binding of this substrate to the catalytic site as compared to the homologous enzymes of other organisms. Aspartate transcarbamoylase of Pyrococcus abyssi exhibits a remarkable stability towards high temperature and pressure.
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A family of diacyltrehaloses isolated from Mycobacterium fortuitum
More LessA trehalose-containing glycolipid was detected in several strains of Mycobacterium fortuitum and characterized as 2,3-di-O-acyltrehalose (DAT) by combined NMR spectroscopy, IR spectroscopy, GLC and GLC-MS. Lipid constituents of the molecule were identified as a mixture of straight-chain (14-18 carbon atoms) and methyl-branched-chain (17-21 carbon atoms) fatty acyl groups. DAT was further fractionated by reverse phase TLC into four fractions that were designated DAT-I-DAT-IV. DAT-I contained 70-75% straight-chain acyl substituents (hexadecanoyl and octadecanoyl predominating) and 25-30% 2-methyl branched substituents (mainly 2-methyl octadecadienoyl). DAT-II was composed of a mixture in which the acyl groups were almost exclusively 2-methyl branched, with 2-methyl octadecadienoyl and 2-methyl octadecen-2-oyl predominating. DAT-III, which was the major isolated fraction, consisted of compounds in which the ratio linear to branched acyl groups varied between 0.8 to 0.9, 2-methyl octadecen-2-oyl, hexadecanoyl and octadecanoyl being the most abundant. Finally, DAT-IV comprised a mixture of DAT molecules containing mostly 2-methyl octadecadienoyl, 2-methyl octadecen-2-oyl, 2-methyl eicosadienoyl and 2-methyl eicosen-2-oyl groups.
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Multiple lactate dehydrogenase activities of the rumen bacterium Selenomonas ruminantium
More LessThe lactate utilizing strain of Selenomonas ruminantium 5934e was found to contain three lactate dehydrogenase (LDH) activities in sonicated cell extracts. One activity, an NAD dependent L-LDH (L-nLDH) was measured at 15-fold greater levels in extracts of cells grown to mid-exponential phase on glucose compared to cells grown to the equivalent growth stage on DL-lactate. A second nLDH activity specific for D-lactate (D-nLDH) was detected at similar levels in both lactate-grown cell extracts and glucose-grown cell extracts. The third activity, an NAD independent DLDH (D-iLDH) was very low in cells grown on glucose but was induced more than 10-fold when DL-lactate was used as the carbon source. The three LDH activities could be separated by gel filtration. Recovery of the activities was low due to the apparent instability of the enzymes at 4 °C, which was most pronounced in the case of the D-iLDH. A Km for lactate of 0.5 mM was estimated for the D-iLDH and this was considerably lower than the values of 45 mM and 70 mM measured for L-nLDH and D-nLDH respectively. It is proposed that the D-iLDH may be largely responsible for the formation of pyruvate in lactate-grown cells of S. ruminantium strain 5934e. Three other lactate utilizing strains of S. ruminantium, HD4, 5521C1 and JW13 exhibited a similar profile of LDH activities to strain 5934e when grown on glucose and DL-lactate.
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Metabolism of pyruvate and glucose by intact cells of Helicobacter pylori studied by 13C NMR spectroscopy
More LessThe metabolic routes of substrate catabolism by intact cells of H. pylori have been investigated by 13C NMR. Real time analyses of metabolic transformations under anaerobic conditions have been obtained with dense cell suspensions incubated with 13C-labelled pyruvate and glucose. In addition, time point studies have been carried out with cells incubated under aerobic conditions. Anaerobically, pyruvate was rapidly metabolized to lactate, ethanol and acetate. In addition, alanine was produced in significant quantities by cells provided with a nitrogen source and the metabolic incorporation of nitrogen from urea was demonstrated. Under aerobic conditions acetate was the major oxidation product from pyruvate; no evidence was obtained for tricarboxylic acid cycle activity. Glucose was metabolized more slowly than pyruvate. Anaerobically, two major products were observed and identified as sorbitol and gluconate by gas chromatography/mass spectrometry. Evidence was obtained for the oxidation of glucose to acetate under aerobic conditions. The fate of the 13C label with glucose substrates labelled in different positions showed that this oxidation takes place via the Entner-Doudoroff pathway.
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- Environmental Microbiology
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Monoclonal antibodies for Streptomyces lividans and their use for immunomagnetic capture of spores from soil
More LessMonoclonal antibodies were produced to Streptomyces lividans spore surface antigens. One particular hybridoma cell line, 43H6, produced a monoclonal antibody that reacted exclusively with Streptomyces cluster group 21 in an enzyme-linked immunosorbent assay (ELISA). Antibody 43H6 was found to be of subclass lgG1, kappa light chain. Western blot (immunoblot) analysis revealed that 43H6 recognized a major outer spore polypeptide of about 37000 Da. The epitope was stably maintained in S. lividans spores over at least seven sporulation cycles on laboratory medium and for at least 14 weeks in sterile soil systems. The species group specificity of antibody 43H6 was exploited in the development of an immunocapture technique for the isolation of streptomycetes from soil. Magnetic beads coated with antibody 43H6 were mixed with soil samples seeded with S. lividans spores. Spore-bead complexes were recovered using magnets. Treatment of beads with blocking agents and the inclusion of detergents in the recovery system lessened non-specific binding of spores to beads and improved recovery. In buffer solutions decreasing the spore concentration increased the recovery values for a fixed bead concentration. At a spore concentration of 5 × 107 ml−1 the recovery was 4.3% whilst at 5 × 102 ml−1 it was 76% for a fixed bead concentration of 0.6 mg ml−1. Using a bead concentration of 2 mg per 10 g soil, approximately 30% of the target spore population of 106 c.f.u. was recovered from sterile soil and 4% from non-sterile soil. This method offers a rapid means of selectively recovering and concentrating Streptomyces spores from soil samples.
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- Genetics And Molecular Biology
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Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase
More LessCitrate synthase catalyses the initial reaction of the citric acid cycle and can therefore be considered as the rate-controlling enzyme for the entry of substrates into the cycle. In Corynebacterium glutamicum, the specific activity of citrate synthase was found to be independent of the growth substrate and of the growth phase. The enzyme was not affected by NADH or 2-oxoglutarate and was only weakly inhibited by ATP (apparent K i = 10 mM). These results suggest that in C. glutamicum neither the formation nor the activity of citrate synthase is subject to significant regulation. The citrate synthase gene, gltA, was isolated, subcloned on plasmid pJC1 and introduced into C. glutamicum. Relative to the wild-type the recombinant strains showed six- to eightfold higher specific citrate synthase activity. The nucleotide sequence of a 3007 bp DNA fragment containing the gltA gene and its flanking regions was determined. The predicted gltA gene product consists of 437 amino acids (M r 48936) and shows up to 49.7% identity with citrate synthase polypeptides from other organisms. Inactivation of the chromosomal gltA gene by gene-directed mutagenesis led to absence of detectable citrate synthase activity and to citrate (or glutamate) auxotrophy, indicating that only one citrate synthase is present in C. glutamicum. Transcriptional analysis by Northern (RNA) hybridization and primer extension experiments revealed that the gltA gene is monocistronic (1.45 kb mRNA) and that its transcription initiates at two consecutive G residues located 121 and 120 bp upstream of the translational start.
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Analysis of a ribose transport operon from Bacillus subtilis
More LessThe csa-15 locus of Bacillus subtilis corresponds to an operon encoding proteins which display features characteristic of the ABC group of transporters. Sequence analysis reveals a very high level of identity to the ribose transport operon of Escherichia coli. This hypothesis is supported by the observation that strains carrying mutagenic insertions in this operon are unable to grow on ribose as sole carbon source. Expression of this operon is directed by a single SigA-type promoter which is negatively regulated by Spo0A during the late-exponential/transition state of the growth cycle. Expression is also subject to catabolite repression and this mode of regulation is dominant to control of expression by Spo0A.
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The Bacillus subtilis lipoprotein LpIA causes cell lysis when expressed in Escherichia coli
More LessA gene called lplA (lipoprotein-like) has been isolated from a genomic library of Bacillus subtilis expressed in Escherichia coli. Clones carrying the IpIA gene were selected by the ability of the colonies to give visible haloes of starch hydrolysis. The cloned fragment contains an open reading frame (ORF) of 1509 bp encoding a protein of 56 kDa. The protein contains a typical N-terminal signal sequence, a putative transmembrane anchor domain and a leucine zipper at the C-terminus. The expression of this protein in E. coli causes cell lysis, only the N-terminal domain of the LpIA protein being responsible for this phenotype. The mechanism of cell lysis is similar to that previously suggested for the expression in E. coli of the lipoproteins encoded by the Streptococcus pneumoniae genes malX and amiA. The protein is modified with palmitic acid when secreted in E. coli, confirming that it is a typical lipoprotein.
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Identification of TlpC, a novel 62 kDa MCP-like protein from Bacillus subtilis
More LessWe report the sequence and characterization of the Bacillus subtilis tlpC gene. tlpC encodes a 61.8 kDa polypeptide (TlpC) which exhibits 30% amino acid identity with the Escherichia coli methyl-accepting chemotaxis proteins (MCPs) and 38% identity with B. subtilis MCPs within the C-terminal domain. The putative methylation sites parallel those of the B. subtilis MCPs, rather than those of the E. coli receptors. TlpC is methylated both in vivo and in vitro although the level of methylation is poor. In addition, the E. coli anti-Trg antibody is shown to cross-react with this membrane protein. Inactivation of the tlpC gene confirms that TlpC is not one of the previously characterized MCPs from B. subtilis. Capillary assays were performed using a variety of chemoeffectors, which included all 20 amino acids, several sugars, and several compounds previously classified as repellents. However, no chemotactic defect was observed for any of the chemoeffectors tested. We suggest that TlpC is similar to an evolutionary intermediate from which the major chemotactic transducers from B. subtilis arose.
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Lytic enzymes associated with defective prophages of Bacillus subtilis: Sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX
More LessProphage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.
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Molecular cloning of two Pseudomonas flagellin genes and basal body structural genes
More LessPseudomonas putida strains PaW8 and PRS2000 produce flagellins with apparent molecular masses of 81 kDa and 50 kDa respectively. Two Tn5 insertion mutants of P. putida PaW8 lacking the ability to bind the flagellin-specific monoclonal antibody MLV1 were isolated. Mutant PaW8-flg2 contained a Tn5 insertion within a 2.6 kb EcoRI fragment of the P. putida chromosome carrying putative basal body genes. DNA and deduced protein sequences suggested the presence on this fragment of two complete genes homologous to flgH and flgl from Salmonella typhimurium. The insertion of Tn5 occurred in the flgH locus and appeared severely to reduce expression of the P. putida flagellin gene. A Tn5-containing fragment of DNA from a second mutant, PaW8-flg1, was cloned and found to contain sequences that hybridized strongly with the Pseudomonas aeruginosa flagellin gene. A 2.3 kb HindIII fragment containing all but 62 bp of the P. putida PaW8 flagellin gene was cloned and used as a probe to identify clones carrying the equivalent gene from P. putida PRS2000. Flagellin genes from both P. putida strains were sequenced and their amino acid sequences deduced. Both flagellins were found to contain conserved amino- and carboxy-terminal regions when compared to other flagellins, with the central region being more variable. The epitope for MLV1 is likely to lie within this central region of P. putida PaW8 flagellin. The deduced molecular mass of P. putida PaW8 flagellin (68 kDa) differed significantly from its apparent molecular mass estimated by PAGE, possibly as a consequence of post-translational modification. This was not the case with flagellin from P. putida PRS2000, where the predicted and apparent molecular masses were similar.
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Population structure of Actinobacillus actinomycetemcomitans: A framework for studies of disease-associated properties
More LessThe Actinobacillus actinomycetemcomitans population consists of a large number of clones among which the ubiquitous leukotoxin gene operon appears very homogeneous. Population genetic analyses performed by multilocus enzyme electrophoresis together with DNA fingerprinting and analyses of genomic DNA restriction fragment length polymorphisms (RFLP) on 97 strains isolated over a period of 45 years revealed that each of the serotypes a, b, c, d and e comprise genetically isolated subpopulations and that successful horizontal transfer of genomic DNA between strains of different serotypes appears to be extremely rare in vivo. In contrast, recombination between strains of the same serotype in general appears to take place in nature. The results provide evidence that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.
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Expression in Escherichia coli of the extracellular basic protease from Dichelobacter nodosus
More LessDichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, secretes a number of extracellular proteases, one of which is highly basic in nature. The gene (bprV) encoding this basic protease, from virulent strain 198, has been cloned and sequenced. Clone pBR3KB contained the complete bprV gene which constitutively expressed an active protease using its own promoter, when cloned in Escherichia coli. However, levels of protease expression were low and unstable when the clone was expressed in liquid culture. A range of E. coli strains were examined for stable expression; strains NH274 and SURE™ were found to be better hosts for stable expression than other commonly used E. coli host strains. Stabilization and enhancement of expression was achieved by deletion of the native promoter region and expression from plasmid promoter or promoters, and by modification of culture conditions. The recombinant protease obtained from E. coli was indistinguishable from the native enzyme in size, activity, isoelectric point and immunological properties.
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Genetic analysis of uidA expression in enterohaemorrhagic Escherichia coli serotype 0157:H7
More LessIsolates of enterohaemorrhagic Escherichia coli serotype 0157:H7 do not exhibit β-glucuronidase (GUD) activity; however, they carry nucleotide sequences for the uidA gene that encodes the GUD enzyme. Polymerase chain reaction analysis using uidA-specific primers confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in E. coli 0157:H7 isolates. DNA sequencing analysis of the regulatory region and the 5′ terminus of the uidA gene of E. coli 0157:H7 showed a base substitution in the putative −10 promotor region and at 93 bases downstream from the initiation codon. Neither base alteration, however, appeared to affect uidA allele gene expression in the 0157:H7 isolates. Immunoblotting of cell extracts with an anti-GUD antibody showed that E. coli 0157:H7 isolates produced an antibody-reactive protein that was homologous in size to E. coli GUD, but no GUD activity was observed in cell-free extracts of these isolates. These results suggest that the antibody-reactive protein produced by E. coli 0157:H7 may be an inactive GUD enzyme. Sequencing of the uidA structural gene in 0157:H7 showed the presence of 18 additional nucleotide base substitutions, but only six altered the amino acid sequence. Also, there were two frame shift mutations, 18 bases apart, that altered the sequence of six consecutive amino acids. These genetic alterations in the uidA structural gene of 0157:H7 may account for the absence of GUD activity in this serotype.
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Cloning and DNA sequence analysis of the region containing attP of the temperate phage ΦAR29 of Prevotella ruminicola AR29
More LessPhage ΦAR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/Hindlll fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal Dral site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage λ. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP. The proposed ΦAR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.
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Nuclear replacement during mating in Armillaria ostoyae (Basidiomycotina)
More LessIn Armillaria ostoyae diploid mycelium may mate with haploid mycelium in a process analogous to dikaryon-monokaryon matings in other basidiomycete fungi. Cultural characteristics and molecular markers were used to study inheritance of nuclear and mitochondrial DNA in four diploid-haploid matings of A. ostoyae. When haploids are mated with diploids, morphological changes from fluffy to flat in the haploid thallus can be used to follow mating progress. Progeny originating from the haploid thallus had mitochondrial haplotypes identical to the haploid parent. Progeny with fluffy colony morphology (putatively haploid) all had nuclear haplotypes identical to the haploid parent. Flat progeny (putatively diploid) had nuclear haplotypes either the same as the diploid parent or a combination of all of the diploid and haploid parent nuclear markers. Diploid-diploid pairings between the parents and flat progeny resulted in somatic incompatibility between genetically unlike diploids and demonstrated that somatic incompatibility is a reliable indicator of nuclear, but not mitochondrial, condition. The data suggest that nuclei of the diploid parent, but not mitochondria, migrate into the haploid thallus and eventually displace the haploid nuclei. In some instances, stable 2N + N dikaryons were maintained.
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Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli
More LessRepression of tryptophanase (tryptophan indole-lyase) by glucose and its non-metabolizable analogue methyl α-glucoside has been studied employing a series of isogenic strains of Escherichia coli lacking cyclic AMP phosphodiesterase and altered for two of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), Enzyme I and Enzyme IIAGIc. Basal activity of tryptophanase was depressed mildly by inclusion of glucose in the growth medium, but inducible tryptophanase synthesis was subject to strong glucose repression in the parental strain, which exhibited normal PTS enzyme activities. Methyl α-glucoside was without effect in this strain. Loss of Enzyme I decreased sensitivity to repression by glucose but enhanced sensitivity to repression by methyl α-glucoside. Loss of Enzyme IIAGIc activity largely abolished repression by methyl α-glucoside but had a less severe effect on glucose repression. The repressive effects of both sugars were fully reversed by inclusion of cyclic AMP in the growth medium. Tryptophan uptake under the same conditions was inhibited weakly by glucose and more strongly by methyl α-glucoside in the parental strain. Inhibition by both sugars was alleviated by partial loss of Enzyme I. Inhibition by methyl α-glucoside appeared to be largely due to energy competition and was not responsible for repression of tryptophanase synthesis. Measurement of net production of cyclic AMP as well as intracellular concentrations of cyclic AMP revealed a good correlation with intensity of repression. The results suggest that while basal tryptophanase synthesis is relatively insensitive to catabolite repression, inducible synthesis is subject to strong repression by two distinct mechanisms, one dependent on enzyme IIAGIc of the PTS and the other independent of this protein. Both mechanisms are attributable to depressed rates of cyclic AMP synthesis. No evidence for a cyclic-AMP-independent mechanism of catabolite repression was obtained.
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Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium
More LessMajor immunogenic polypeptides (21, 32, 40, 46, 58, 85 and 160 kDa) of Cowdria ruminantium were identified by immunoprecipitation and immunoblotting. A pUC13 library of C. ruminantium genomic DNA was screened with hyperimmune sheep serum to identify Escherichia coli colonies which expressed genes encoding these immunogenic proteins. A recombinant E. coli colony, F5.2, was identified containing plasmid insert DNA of 2773 bp. The cloned DNA insert contained two long open reading frames (ORFs) of 627 bp (complete) and 831 bp (incomplete), both potentially encoding proteins containing an N-terminal signal peptide. Deletion experiments suggested that the hyperimmune sheep serum recognized a protein that was encoded by the 627 bp ORF. The 627 bp ORF was amplified by polymerase chain reaction (PCR), subcloned and expressed to a high level in E. coli. A sheep antiserum made to the expressed recombinant fusion protein recognized a 21 kDa protein of all strains of C. ruminantium tested, confirming that the 627 bp ORF encodes a native 21 kDa protein in C. ruminantium. Similarly, the recombinant protein was recognized by all sera tested from heartwater-infected animals. The antigenic conservation of the 21 kDa protein and its immunogenic nature are reasons for further testing of this recombinant protein in subunit diagnostic tests.
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Nucleotide sequence and characterization of the Rhodobacter sphaeroides glnB and glnA genes
More LessThe glnA gene of Rhodobacter sphaeroides encoding glutamine synthetase (GS) has been cloned and sequenced. Molecular analysis revealed that there is a glnB gene upstream of glnA, in a single glnBA operon. A putative glnAp1-type promoter sequence, a consensus ntrC gene product binding site and a consensus upstream activator sequence were detected upstream of the glnB gene. The deduced amino acid sequences of the GS and GlnB proteins of R. sphaeroides showed strong homology with the same proteins from other Gram-negative bacteria. The sequence of the glnA gene isolated from glutamine auxotroph Gln83 was also determined. The glnA83 mutation was shown to result in premature termination of GS synthesis and formation of a 17 kDa C-truncated GS which could be complemented by a 5′-truncated glnA gene which encodes a 30 kDa N-truncated GS. This phenomenon is characteristic for interallelic complementation.
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- Pathogenicity And Medical Microbiology
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Identification and characterization of a putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis
More LessA putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis was isolated from a lambda gt11 genomic expression library by screening with serum from a naturally infected sheep. The gene was contained in two overlapping clones, which were shown by antibody elution to encode a protein of 34 kDa in M. a. paratuberculosis. The clones were sequenced and database searches detected a motif identical to the active serine site in trypsin, and 30% homology to the putative serine proteases (HtrA proteins) of Escherichia coli, Salmonella typhimurium, Brucella abortus and Rochalimaea henselae.
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Mycobacterium leprae isolates from different sources have identical sequences of the spacer region between the 16S and 23S ribosomal RNA genes
More LessTo test for genotypic variations between different isolates of Mycobacterium leprae, the causative agent of leprosy, the 282 bp spacer region between the 16S and 23S rRNA genes was amplified using PCR, and submitted to single-strand conformation polymorphism (SSCP) analysis. The procedure was optimized using four modified spacer fragments, containing mutations at one, three, four and six positions, respectively. Seventy-five M. leprae isolates from different sources, including isolates from leprosy patients, healthy individuals, armadillos and mouse footpads were identical in the SSCP analysis. DNA sequencing and restriction enzyme analysis performed on four and 40 samples, respectively, confirmed the results obtained with SSCP analysis.
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Cytokine gene expression in the lungs of BALB/c mice during primary and secondary intranasal infection with Mycoplasma pneumoniae
More LessCytokine gene expression was determined in vivo in the lungs and spleens of Mycoplasma pneumoniae-infected BALB/c mice by means of qualitative and semiquantitative PCR-mediated mRNA amplification. During the acute phase of both primary and secondary infections, cytokines commonly associated with innate resistance, TNFα, IFNγ, IL-1β and IL-6, were expressed. In contrast, early expression of the genes for IL-2 and IL-2 receptor was detected only during reinfection. Expression was greater in the lungs than in the spleen, attesting to the rapid accumulation of lymphocytes at the infected site. Interestingly, IL-2 mRNA expression declined rapidly and was no longer detectable after 24 h, whereas IL-10 mRNA levels rose sharply during the same period. During reinfection, mRNAs for TNFα and IL-6 were 10-fold and for IFNγ about 50-fold higher than during primary challenge. The results suggest that the pathogenesis of M. pneumoniae diseases may be associated with elevated expression of proinflammatory cytokines.
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- Physiology And Growth
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Activity of the plasma membrane H+-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae
The role of membrane integrity and the membrane ATPase in the mechanism of thermotolerance in Saccharomyces cerevisiae was investigated. The resistance to lethal heat of a mutant strain with reduced expression of the membrane ATPase was significantly less than that of the wild-type parent. However, prior exposure to sub-lethal temperatures resulted in the induction of similar levels of thermotolerance in the mutant compared to the parent strain, suggesting that the mechanism of sub-lethal heat-induced thermotolerance is independent of ATPase activity. Supporting this, exposure to sub-lethal heat stress did not result in increased levels of glucose-induced acid efflux at lethal temperatures and there was little correlation between levels of acid efflux and levels of heat resistance. ATPase activity in crude membrane preparations from sub-lethally heat-stressed cells was similar to that in preparations from unstressed cells. Study of net acid flux during heating revealed that pre-stressed cells were able to protect the proton gradient for longer. This may confer an ‘advantage’ to these cells that results in increased thermotolerance. This was supported by the observation that prior exposure to sub-lethal heat resulted in a transient protection against the large increase in membrane permeability that occurs at lethal temperatures. However, no protection against the large drop in intracellular pH was detected. Sub-lethal heat-induced protection of membrane integrity also occurred to the same extent in the reduced-expression membrane ATPase mutant, further implying that the mechanism of induced thermotolerance is independent of ATPase activity. To conclude, although the membrane ATPase is essential for basal heat resistance, thermotolerance induced by prior exposure to stress is largely conferred by a mechanism that is independent of the enzyme.
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Decrease in glycolytic flux in Saccharomyces cerevisiae cdc35-1 cells at restrictive temperature correlates with a decrease in glucose transport
More LessThe glycolytic flux was investigated in the thermosensitive Saccharomyces cerevisiae adenylate cyclase mutant cdc35-1. Directly after a shift to restrictive temperature, the specific CO2 production rate increased from about 250 nmol min−1 (mg protein)−1 to more than 400 nmol min−1 (mg protein)−1, but then the CO2 production gradually fell to about 70 nmol min−1 (mg protein)−1 after 5 h. O2 consumption at restrictive temperature continued at more or less the same rate as at permissive temperature. The temperature shift in the mutant resulted in an increase in the estimated intracellular cAMP concentration from about 1.1 μM to 1.8 μM. This indicates that high cAMP levels are not sufficient for cell cycle progression and high glycolytic activity. The decrease in glycolytic activity at restrictive temperature was not paralleled by a similar decrease in the specific activity of any of the glycolytic enzymes, but correlated with a decrease in hexose transport. A drop in intracellular concentrations of the early metabolites of glycolysis further indicated a defect in transport at restrictive temperature. Our data suggest that glucose transport has a high control on glycolytic flux.
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Volume regulation in Spiroplasma floricola: Evidence that Na+ is extruded by a Na+/H+ antiporter
More LessMarked cell swelling followed by lysis was observed when Spiroplasma floricola cells were incubated in iso-osmotic solutions of NaCl, KCI, choline chloride or sorbitol in the absence of an energy source. In the presence of an energy source the cells did not swell suggesting that S. floricola relies on an energy-dependent mechanism(s) for cell volume regulation. An ammonium chloride dilution procedure was utilized to generate a pH gradient (inside acid) across the cell membrane of S. floricola cells. The addition of NaCl resulted in an intracellular alkalization suggesting the presence of a Na+/H+ exchange activity. In 22Na+-loaded cells, glucose-dependent 22Na+ extrusion was observed at acidic pH in both the presence and absence of Na+ ions. The extrusion was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) and partially inhibited by dicyclohexylcarbodiimide (DCCD, 100 μM) indicating that in S. floricola, Na+ movement is driven by the electrochemical gradient of H+ via a Na+/H+ antiporter. The specific ATPase activity of S. floricola membranes was at least twofold higher than that described in other mollicutes. Activity was Mg2+-dependent over the pH range (6.5-8.5) tested, but was very little affected by Na+ (up to 100 mM). DCCD (25 μM) markedly inhibited both membrane-bound and solubilized ATPase activity, whereas orthovanadate (50 μM) had only a small inhibitory effect. The properties of the enzyme are consistent with a F0F1-ATPase. It is suggested that the enzyme operates in the direction of hydrolysing ATP formed by glycolysis leading to the generation of a δpH, which is the major driving force for the Na+/H+ antiport activity.
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Response of Rhizobium fredii P220 to osmotic shock: Interrelationships between K+, Mg2+, glutamate and homospermidine
More LessThe response of Rhizobium fredii P220, a salt-tolerant strain of soybean rhizobia, to osmotic shock was investigated by using non-growing washed cells. Rapid changes in K+, Mg2+, glutamate and homospermidine were observed in strain P220 cells subjected to sudden changes in the osmolarity of incubation buffer. Osmotic upshock resulted in elevation of cellular K+ and glutamate, and reduction in cellular homospermidine and Mg2+. When the cells were transferred to upshock buffer lacking K+, the reduction in Mg2+ was totally blocked, but the elevation of glutamate and the reduction in homospermidine were only partially repressed. Osmotic downshock resulted in the opposite phenomenon: There was an elevation of homospermidine and Mg2+, and a rapid fall of K+ and glutamate. When the cells were transferred to downshock buffer lacking Mg2+, the elevation of homospermidine was partially repressed, but the decrease in K+ and glutamate was not repressed at all. Lowering of the cellular K+ by treatment with ionophores nigericin and monensin resulted in a slight decrease in glutamate and a slight increase in homospermidine and Mg2+, possibly due to a pH effect caused by the K+-H+ exchange. Raising the cellular Mg2+ content by treatment with ionophore A23187 brought about an increase in homospermidine. The homospermidine content of Mg2+-deficient cells grown with low-Mg2+ medium reduced to 35% of those grown with the basal medium. These results indicate that in R. fredii, K+ strictly controls Mg2+ flux during osmotic shock whereas the reverse is not true, and that glutamate and homospermidine essentially escape direct control by K+. We also suggest that Mg2+, which has no effect on the pool size of glutamate, is one of the factors which regulate homospermidine content in rhizobial cells.
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Autoregulation of nitrogenase expression in Klebsiella pneumoniae
More LessAn investigation into the influence of N2 on the expression of Klebsiella pneumoniae nitrogenase has led to a reassessment of the role of the nitrogenase MoFe protein in autoregulation. Anaerobic derepression of nitrogenase (C2H2-reducing) activity, of NifD and K polypeptides, and of nifH-lac expression, following the removal of excess NH+ 4, were greater under N2 than Ar. This enhancement occurred in Nif+ but not in Nif− strains, and in Nif+ strains was prevented by C2H2, an inhibitor of N2 fixation. Thus N2 fixation is important for maintaining derepression. Derepression of nifH-lac under Ar in various Nif+ and Nif− strains (including NifH−, NifD−, NifB− and NifL− mutants) and of wild-type lac under N2 or Ar in a Nif+ strain were measured to investigate the regulation. The mechanism regulating the enhancement under N2 neither involved the MoFe protein of nitrogenase, as proposed by Dixon et al. (1980, Nature 286, 128-132), nor the nifL product, but was probably due to a general upgrading of the N status. Moreover, during batch growth limited by a non-repressing fixed N source, the levels of nifH-lac expression in the Nif+ and Nif− strains suggested that the nifH gene product (or Fe protein) may have a positive autoregulatory function.
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Growth and regulation of enzyme synthesis in the nitrilotriacetic acid (NTA)-degrading bacterium Chelatobacter heintzii ATCC 29600
More LessIn the aerobic bacterium Chelatobacter heintzii, growth and regulation of enzymes involved in nitrilotriacetic acid (NTA) degradation have been investigated in chemostat culture during cultivation with glucose, NTA or mixtures thereof. In batch culture μmax with NTA was 0.18 h−1 and with glucose 0.22 h−1. Growth yields for both substrates were reduced at low dilution rates. During growth with NTA specific activity of the NTA monooxygenase (NTA-MO) exhibited a maximum at D = 0.03 h−1 and gradually decreased with increasing dilution rates. In glucose-grown cells the specific activity as well as immunologically detectable NTA-MO protein was always close to the detection limit. During cultivation with different mixtures of NTA and glucose at a dilution rate of 0.06 h−1, both substrates were utilized simultaneously, irrespective of the NTA/glucose ratio and the presence of excess ammonia. Synthesis of both NTA-MO and iminodiacetic acid dehydrogenase became induced when NTA contributed to more than approximately 1-3% of the total carbon in the substrate mixture supplied. However, NTA was also degraded when the proportion of NTA in the mixture was lower than 1%, which is consistent with the low constitutive level of expression for NTA-MO observed. Results are discussed with respect to NTA biodegradation during sewage treatment and in ecosystems.
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Inhibition of lipid biosynthesis induces the expression of the pspA gene
More LessTreatment of Escherichia coli with diazaborine strongly induces the synthesis of a 28 kDa protein which is associated with the cytoplasmic membrane. The partial amino acid sequence proved that this protein is identical to the phage shock protein PspA. The kinetics of the expression of the pspA gene were determined in an E. coli strain which carried a pspA-lacZ fusion in the chromosome. PspA synthesis is independent of the growth phase. It is, however, strongly induced when fatty acid biosynthesis is inhibited by diazaborine or cerulenin. Treatment with either compound also causes dose-dependent inhibition of phospholipid biosynthesis whose degree correlates with the induction of PspA. Another cause of induction of PspA synthesis is treatment of E. coli with globomycin, which is an inhibitor of the processing of lipoproteins.
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Metabolic and energetic aspects of the growth response of Streptococcus rattus to environmental acidification in anaerobic continuous culture
More LessStreptococcus rattus, a serotype b strain of mutans streptococci, was grown in an anaerobic glucose-limited chemostat. The molar growth yield of glucose [Y glucose, g dry wt (mol glucose)−1] together with the maximum growth yields (Y max) and maintenance coefficients for glucose utilization and calculated ATP generation were estimated as a function of pH. When the pH was lowered from 7.0 to 5.0, Y glucose decreased, with a concomitant gradual change in the composition of the end product from a mixture of formate, acetate and ethanol to one mostly of lactate. Whereas the Y max for glucose decreased without any change in the Y max for ATP on acidification, both of the maintenance coefficients markedly increased. Kinetic and immunochemical examinations indicated the presence of an F1F0-type proton-translocating ATPase in the membrane fraction prepared from bacterial cells grown under acidic conditions; no detectable level of the enzyme was found in cells grown at neutral pH. However, when incubated with glucose under non-growing conditions, these acid-adapted and unadapted cells showed an insignificant difference in the ability to maintain the intracellular pH alkaline relative to the acidic environments. These results suggest that the organism responds and adapts to environmental acidification by sacrificing some energy cost in terms of both the efficiency of glucose utilization to generate ATP and the extra maintenance required to continue biomass production as efficiently as under neutral pH.
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Isolation and characterization of a strain of Rhodobacter sulfidophilus: A bacterium which grows autotrophically with dimethylsulphide as electron donor
More LessA marine photosynthetic bacterium (strain SH1) was isolated after enrichment under phototrophic conditions in media containing dimethylsulphide (DMS) and bicarbonate (HCO− 3) as potential carbon sources. Analysis of culture medium using nuclear magnetic resonance spectrometry showed that during phototrophic and chemotrophic growth of strain SH1 on DMS/HCO− 3 dimethylsulphoxide (DMSO) was produced from DMS. These results indicate that strain SH1 grew autotrophically with DMS serving as an electron donor in photosynthesis and respiration, but not as a carbon source. Biochemical characterization and 16S rRNA analysis indicated that the isolate was a strain of Rhodobacter sulfidophilus. An assay for the enzyme catalysing the oxidation of DMS (DMS:acceptor oxidoreductase) was developed by measuring electron transfer from DMS to 2,6-dichlorophenolindophenol (DCPIP). This reaction was dependent on phenazine ethosulphate to mediate electron transfer from DMS:acceptor oxidoreductase to DCPIP. DMS:acceptor oxidoreductase was found to have a periplasmic location in strain SH1 as was a reduced methylviologen: DMSO oxidoreductase activity. Zymogram staining patterns of periplasmic fractions indicated that DMS: acceptor oxidoreductase and DMSO reductase were distinct enzymes. This was confirmed by resolution of the two activities by gel filtration.
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Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene
More LessDefence mechanisms of three Pseudomonas putida strains growing in the presence of toluene up to 50%
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Biochemical diversity within the ‘Mycoplasma mycoides cluster‘
More LessThe metabolism of 51 strains within the ‘Mycoplasma mycoides cluster’ was investigated by measuring oxygen uptake following the addition of organic substrates to washed cell suspensions. There were extensive differences between strains in the range of substrates utilized, the relative rates of oxidation and the observed saturation constants for substrates, which ranged from a few μM to several mM. M. mycoides subsp. capri and M. mycoides subsp. mycoides LC (large colony) strains were diverse and could not be distinguished by substrate utilization patterns. However, there were consistent differences in the patterns of substrate utilization between other groups of the M. mycoides cluster, suggesting that these patterns may be useful in identification. In particular, SC (small colony) strains of M. mycoides subsp. mycoides were distinguished by their inability to oxidize maltose, trehalose and (at low concentrations) mannose and glucosamine. Surprisingly, the type strain, M. F38, of Mycoplasma capricolum subsp. capripneumoniae and two further isolates differed from all other strains in that they did not oxidize glucose or other sugars. They did, however, oxidize pyruvate, lactate and 2-oxobutyrate at high rates. The marked metabolic differences between these strains and M. capricolum subsp. capricolum strains is in contrast to the genetic evidence that was used to support the designation of the M. F38 group as a subspecies of M. capricolum.
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Kinetic characterization of sporulation in Streptomyces albidoflavus SMF301 during submerged culture
More LessWe report the first quantitative analysis of the relationship between environmental changes and sporulation of a streptomycete, Streptomyces albidoflavus SMF301, in submerged culture. A chemically defined medium was constructed for sporulation, over 109 spores ml−1 being formed in the submerged batch culture. Kinetic parameters calculated from batch and chemostat cultures showed that specific submerged spore formation rate (qspo) was inversely related to the specific mycelial growth rate (μ). The optimum growth rate for submerged spore formation was 0.05 h−1, when the maximum value of qspo was 1.0 × 106 spores g−1 h−1. The turnover rate of biomass at maximum growth yield was 0.029 h−1 when 5.6 × 106 spores were formed from 1 g mycelium. The present quantitative analysis of submerged spore formation using a controlled system opens the way for biochemical and molecular biological studies related to the morphological differentiation of Streptomyces spp.
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Diazotrophic synchronous growth of a marine unicellular cyanobacterium, Synechococcus sp. strain Miami BG 043511, under aerobic and microaerobic/anaerobic conditions
More LessThe growth attributes of an aerobic nitrogen-fixing Synechococcus strain, Miami BG 043511, under aerobic and microaerobic/anaerobic conditions were examined using conventional batch and synchronous culture methods. Generation times of this strain, estimated from the increase in cell density under aerobic and anaerobic batch culture conditions, were 19-23 h and 15-19 h at 30 °C, respectively. It seems, therefore, that atmospheric oxygen did not seriously affect diazotrophic growth in this strain. Under a periodic light-dark regime, cells grew synchronously even under microaerobic/anaerobic conditions. When the aerobic culture entered the light period, a peak of photosynthetic activity was followed by a peak of nitrogenase activity. In contrast, a peak of nitrogenase activity preceded a peak of photosynthetic activity under microaerobic/anaerobic conditions. In both cases, however, cell division was observed at or just after the peak of photosynthetic activity. The difference in the timing of the appearance of nitrogenase activity in microaerobic/anaerobic cultures was ascribed to the inability of cells to generate sufficient ATP under anaerobic dark conditions. Periodic changes in cellular carbohydrate content, associated with the periodic appearances of photosynthetic and nitrogen-fixing activities, were observed under both aerobic and microaerobic/anaerobic conditions. Cellular carbohydrate content increased from 10% to 60% of cell dry weight during the phase of photosynthesis under aerobic conditions, while it reached only 40% under microaerobic/anaerobic conditions. The amount of reserve polysaccharides required to support nitrogen fixation was larger in aerobic cultures than in microaerobic/anaerobic cultures.
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Osmoresistance of spores from Bacillus subtilis and the effect of ssp mutations
More LessSpores of Bacillus subtilis show similar plating efficiency on media with or without 1.5 M NaCl. In contrast, vegetative cells are osmosensitive unless the stationary phase has been reached. In the present work, loss of heat and osmotic resisitance during germination was studied. Their kinetics and sensitivity to protein synthesis inhibition were different: heat resistance was lost first and even in the presence of chloramphenicol, whereas loss of osmotolerance occurred later and was inhibited in the presence of this antibiotic. The influence of spore-associated small acid-soluble proteins (SASPs) on spore osmotolerance was investigated using ssp mutants: all produced spores which germinated poorly and were sensitivei to osmotic strength. SASP-E deficiency was particularly significant. Spore osmotolerance was largely restored in complementation assays performed with cloned ssp genes. It is possible that germination-associated degradation of SASP proteins provides osmotically significant levels of amino acids (especially glutamate).
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- Systematics
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Evolutionary affiliation of the marine nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067, derived by 16S ribosomal RNA sequence analysis
More LessThe 16S rRNA sequence of Trichodesmium sp. strain NIBB 1067 was determined and used for the construction of a distance tree and bootstrap analysis. The tree shows that, among the available cyanobacterial 16S rRNA sequences, Trichodesmium NIBB 1067 has Oscillatoria PCC 7515 as its closest relative, presenting 94.9% of sequence similarity with the latter strain. This is in contrast to a difference of 9 mol% G + C in mean genomic DNA base composition between the two organisms. Nevertheless, the genotypic heterogeneity presented by a number of strains assigned to the genus Oscillatoria hinders a taxonomic decision on the separate existence of the genera Trichodesmium and Oscillatoria. The sequence of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes was also determined, as a possible marker to study inter- and intraspecific variability. The ITS contains the genes coding for tRNAlle and tRNAAla and its total length is 547 nucleotides. In six out of eight sequenced clones, there is a duplication of 29 nucleotides, surrounding the 5′ end of the tRNAlle.
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Phylogenetic diversity in the genus Bacillus and comparative ribosomal protein AT-L30 analyses of the genus Thermoactinomyces and relatives
More LessThe ribosomal L30 proteins from strains of 27 species belonging to the genera Bacillus, Escherichia, Staphylococcus, Lactobacillus, Leuconostoc and Thermoactinomyces were analysed, together with AT-L30 proteins from selected actinomycetes. The results of partial amino acid sequencing of L30 preparations revealed that the members of the genera Escherichia, Staphylococcus and Thermoactinomyces were homogeneous within each genus. In contrast, phylogenetic diversity existed in the genus Bacillus, which contained at least four clusters. One cluster that contained Bacillus subtilis and Bacillus stearothermophilus was more closely related to the genus Staphylococcus than to members of the other three Bacillus clusters. Members of the genus Thermoactinomyces were most closely related to the ‘Bacillus subtilis cluster’, but less related to the other three Bacillus clusters. A distant phylogenetic relationship was detected between the genus Thermoactinomyces and its morphological relative, Thermomonospora.
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