- Volume 134, Issue 3, 1988
Volume 134, Issue 3, 1988
- Biochemistry
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NADPH Generation in Aspergillus nidulans: Is the Mannitol Cycle Involved?
More LessA cyclic pathway of NADPH generation involving interconversion of mannitol and fructose has been proposed to occur in fungi. In Aspergillus nidulans three enzymes of this proposed mannitol cycle (hexokinase, NADP-mannitol dehydrogenase and mannitol-1-phosphate phosphatase) were shown to be localized exclusively in the cytosol. Two isoenzymes of the fourth enzyme (mannitol-1-phosphate dehydrogenase) were detected and shown to be localized respectively in the mitochondrion and the cytosol. The mitochondrial isoenzyme appeared to be present on the outer face of the inner mitochondrial membrane. No evidence was found for a coordinated change in the maximal activities of the enzymes of the proposed mannitol cycle in extracts prepared from mycelia grown on six different carbon, and three different nitrogen sources nor for any increase in these activities induced by growth on NO3 −. Studies of this type in which other NADP-linked dehydrogenases were measured showed that for most carbon sources tested growth on NO3 − increased the maximal activity of NADP-isocitrate dehydrogenase as well as that of glucose-6-phosphate and 6-phosphogluconate dehydrogenases but had little effect on the maximal activity of NADP-malate dehydrogenase (decarboxylating). Our studies provide no support for the operation of the mannitol cycle, or for the proposed role of this cycle in NADPH generation in A. nidulans.
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Biosynthetic Origin of Aflatoxin G1: Confirmation of Sterigmatocystin and Lack of Confirmation of Aflatoxin B1 As Precursors
More LessThe origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.
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Levels of Polyamines, Glutathione and Glutathione-Spermidine Conjugates during Growth of the Insect Trypanosomatid Crithidia fasciculata
More LessLevels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12·1 to 17·4 nmol per 108 cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 108 cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 108 cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.
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Mechanisms of Sugar Transport in the Rumen Bacterium Selenomonas ruminantium
More LessToluene-treated cells of Selenomonas ruminantium HD4 used phosphoenolpyruvate (PEP) to phosphorylate glucose and sucrose. Glucose activity was constitutive, while the phosphorylation of sucrose was inducible. Competition experiments indicated that separate phosphotransferase (PTS) enzymes II were present for glucose and sucrose, but it appeared that maltose was hydrolysed by an inducible extracellular maltase and then transported by the glucose PTS. S. ruminantium HD4 grew more slowly on maltose than glucose or sucrose and the specific activity of maltase was rate limiting. The maltase was competitively inhibited by glucose and sucrose. Xylose was not phosphorylated by PEP or ATP, and its uptake was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), and by chlorhexidine diacetate. The absence of PEP-dependent phosphorylation and the effects of CCCP suggested that xylose was transported by an active transport mechanism.
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- Biotechnology
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The Effect of Detergents on Amino Acid Liberation by the N2-fixing Cyanobacterium Anabaena variabilis and 6-Fluorotryptophan-resistant Mutant Strains
More LessThe effects of the polyoxyethylene stearate detergents MYRJ 45 and MYRJ 52 on FT-9, an amino-acid-liberating mutant strain of Anabaena variabilis, and on the parent strain were investigated. Both strains were capable of growth in the presence of these detergents, which induced the liberation of amino acids by the parent strain and increased the liberation of amino acids by the mutant strain. Some changes in the spectrum of amino acids liberated by the mutant strain were observed in the presence of detergents, notably the liberation of tryptophan, which was not detected in the absence of detergents.
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- Development And Structure
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Isolation and Characterization of Cell Wall Components of the Unicellular Cyanobacterium Synechococcus sp. PCC 6307
More LessCell walls and outer membranes, free of thylakoids and cytoplasmic membranes, were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 6307. Electron microscopy revealed C-shaped cell wall fragments, which were partially converted to outer membrane vesicles after removal of the peptidoglycan by lysozyme digestion. The major constituents of the outer membrane were proteins and lipopolysaccharide, while lipids and carotenoids were minor components. The polypeptide patterns of the outer membranes were dominated by two major proteins (M r 52000 and 54000). Five strongly polar lipids (unidentified), free fatty acids and small amounts of sulpholipid were detected in extracts of partially purified cell wall fractions, but monogalactosyldiglyceride and digalactosyldiglyceride were not found. The peptidoglycan layer (10 nm thick) was also isolated. Its chemical composition indicated an Al γ-type structure. The degree of cross-linkage was 57%. A polysaccharide, consisting of fucose, mannose, galactose and glucose, was bound to the peptidoglycan, most likely via muramic acid 6-phosphate.
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Isolation and Chemical Analysis of the Sheaths of the Filamentous Cyanobacteria Calothrix parietina and C. scopulorum
More LessThe sheaths of two species of cyanobacteria, Calothrix parietina (two strains) and C. scopulorum (one strain), were studied. Their fine structure showed osmiophilic fibres running parallel to the cell surface. Enriched sheath fractions were obtained from both species in high yields in the phenol/water interphase after hot phenol/water extraction of cell homogenates. The purified sheaths of the two species had similar chemical compositions, with about 50% (of sheath dry weight) neutral sugars (galactose, glucose, mannose, xylose, arabinose, rhamnose, fucose, a 2-O-methylhexose and an unidentified 2-O-methyl sugar), 5% amino acids and small amounts of glucosamine and galacturonic acid. The sheath composition was independent of changes in the iron or phosphate content of the culture medium. The sheath was insoluble in water, and its chemical composition remained essentially unchanged on application of drastic extraction methods, including boiling in 2% (w/v) SDS. The sheath bound heavy metals (up to at least 0.7% of sheath dry weight) with the effectiveness (measured as absolute quantities) Fe > Zn > Cu > Ni > Mn > Mo > Co. Ni, Cu, Zn and Fe were highly enriched relative to their concentration in the culture medium. The concentration factors for Mo. Mn, Ca, Na and K were low.
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- Ecology
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Toxigenicity of Some Fusaria Associated with Plant and Human Diseases in the Malaysian Peninsula
More LessIn the course of a plant disease survey of the Malaysian Peninsula (Malaysia comprises the Malaysian Peninsula, Sabah and Sarawak) during the period 1981-1986, more than 1000 isolates of Fusarium were obtained from diseased plants and seeds. Two further isolates were obtained from patients admitted to hospitals in the same area. The occurrences of F. proliferatum, F. nygamai and F. longipes are new records for the Malaysian Peninsula and the association of F. solani and F. oxysporum var. redolens with human diseases does not seem to have been reported previously. Ten representative species which could be classified into seven sections of the genus were selected for studies of their toxigenicity in liquid cultures and/or on rice. Crude toxin preparations from culture filtrates or extracts of the inoculated rice were tested for toxicity to brine shrimp larvae and tobacco mesophyll protoplasts. The protoplasts were more sensitive than the brine shrimp larvae to the toxin preparations, except those from the isolates of F. solani and F. oxysporum var. redolens obtained from either humans or tobacco. The toxicity of the preparations from rice cultures per g rice was always greater than the toxicity per ml of culture filtrates from cultures grown on Czapek-Dox broth, Czapek-Dox supplemented with 1% (w/v) peptone or Czapek-Dox supplemented with 5% (w/v) tobacco extract. The activity of all toxin preparations was stable to heat. It is concluded that the occurrence of toxigenic species of Fusarium in the Malaysian Peninsula is widespread and that they may pose a serious threat to the health of human, animal and plant populations.
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Selective Isolation of Mycobacteria from Soil: a Statistical Analysis Approach
More LessWe compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: Lwenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 g cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.
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- Genetics And Molecular Biology
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Preferential Uptake of Restriction Fragments from a Gonococcal Cryptic Plasmid by Competent Neisseria gonorrhoeae
More LessFactors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4·2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4·2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.
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Distribution of Plasmid Sequences in Avian and Mammalian Strains of Chlamydia psittaci
More LessPlasmid DNA from an avian strain of Chlamydia psittaci was purified and estimated to be 7·9 kb in size using restriction endonuclease analysis. A 5·9 kb fragment of this plasmid was cloned, mapped and used to screen a range of chlamydial strains. Hybridizing DNA was absent from ovine abortion and arthritis isolates and also from the Cal 10 strain but related sequences were detected in C. psittaci strains of feline pneumonitis, guinea-pig inclusion conjunctivitis, ovine conjunctivitis and C. trachomatis serovar L2. The plasmid DNA from the feline strain was shown to have a distinct restriction endonuclease profile. Similar plasmid sequences were detected in all avian isolates tested: thus the clone may have a useful diagnostic role for the detection of the pathogen in its natural host and in zoonotic episodes.
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Cloning, Expression in Escherichia coli and Nucleotide Sequence of a Tetracycline-resistance Gene from Streptomyces rimosus
More LessDeterminants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9·5 and a 7·5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14·0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2·45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of λpL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (M r 35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.
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Accumulation of LamB-LacZ Hybrid Proteins in Intracytoplasmic Membrane-like Structures in Escherichia coli K12
More LessThe subcellular location of LamB-LacZ hybrid proteins in the Escherichia coli K12 strains pop3234 and pop3299 was investigated by immunocytochemical detection and protease-accessibility experiments. Induction of the synthesis of the hybrid proteins resulted in the appearance of membrane-like structures within the cytoplasm of the cells. Labelling of ultrathin cryosections of the cells with anti-β-galactosidase or anti-LamB protein serum and protein-A-gold complexes revealed that the hybrid proteins were associated with these membrane-like structures or accumulated within the cytoplasm. Protease-accessibility experiments confirmed this localization. Moreover, when low quantities of hybrid proteins were produced, i.e. in uninduced pop3234 cells or in induced pop3299 cells, the hybrid proteins were accessible to trypsin from the periplasmic side of the inner membrane, leaving protected fragments with an apparent M r of 83000. Apparently, these hybrid proteins are partly translocated through the inner membrane, resulting in membrane-spanning forms of the proteins.
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Construction of a Shuttle Vector for Inducible Gene Expression in Escherichia coli and Bacillus subtilis
More LessThe construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The laclq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.
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Effects of Oxygen Levels on the Transcription of nif and gln Genes in Bradyrhizobium japonicum
More LessThe transciption of genes that function in N2 fixation (nif) and nitrogen assimilation (glnII) in Bradyrhizobium japonicum is coordinately induced in response to O2 limitation as well as symbiotic development. We have determined the relative steady-state mRNA levels for the nifH, nifDK and glnII transcription units in bradyrhizobial cells grown under a variety of levels of aerobiosis and in cells isolated from soybean root nodules. All three transcripts are found in cells grown in a rich medium sparged with O2 concentrations of 5 % (v/v) or less. This expression is qualitatively similar to that observed for Bradyrhizobium during symbiotic development. Potential physiological mechanisms for the coordinate control of these genes are discussed.
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Interphylum Protoplast Fusion and Genetic Recombination of the Algae Porphyridium cruentum and Dunaliella spp
More LessTrue protoplasts of the red alga Porphyridium cruentum and the halophilic green algae Dunaliella bardawil and Dunaliella salina were induced by treatment with pectinase and cellulysin. Protoplasts of P. cruentum and the Dunaliella spp. were fused by polyethylene glycol treatment and then allowed to regenerate on selection media. Fusants that were essentially Porphyridium in nature were generated between P. cruentum and D. bardawil and D. salina; these tolerated a salinity of up to 3·5 m for the former and 2·5 m for the latter, indicating that they were hybrid strains, acquiring the osmotolerance from the Dunaliella spp. A Dunaliella-like clone was isolated from protoplast fusion between P. cruentum and D. salina, with altered antibiotic sensitivity. The acquired resistance to penicillin and erythromycin appears to be the result of a genetic transfer from P. cruentum.
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Glycerol Uptake Mutants of the Hyphal Fungus Aspergillus nidulans
More LessA new class of glycerol non-utilizing mutants, designated glcC, has been isolated. The glcC gene was mapped in linkage group VI and mutants were found to complement the reference strains glcA1 (linkage group V) and glcB33 (linkage group I) in diploids. The new mutants were unable to grow on glycerol. However, in contrast to the glcA and glcB phenotype these mutants did grow well on dihydroxyacetone and d-galacturonate. By in vivo 13C NMR spectroscopy it was shown that the glcC mutant did not take up glycerol but did take up dihydroxyacetone. The latter substrate was converted intracellularly into glycerol which was then catabolized as normal.
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The Nucleotide Sequence of Staphylococcus aureus Plasmid pT48 Conferring Inducible Macrolide-Lincosamide-Streptogramin B Resistance and Comparison with Similar Plasmids Expressing Constitutive Resistance
More LessThe complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase.
Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.
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DNA Sequencing of the eta Gene Coding for Staphylococcal Exfoliative Toxin Serotype A
More LessWe report the nucleotide sequence of a 1·45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.
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Construction of cys: lac Gene Fusions in Escherichia coli and Their Use in the Isolation of Constitutive cysBc Mutants
More LessOperon fusions of the lacZ gene to two different genes of the cysteine regulon controlled by the cysB regulatory protein were isolated. The fusion strains were used for selection of cysB constitutive mutants. Three cysBc alleles have been characterized and cloned into multicopy plasmids.
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- Pathogenicity And Medical Microbiology
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Lipopolysaccharide Masking of Gonococcal Outer-membrane Proteins Modulates Binding of Bactericidal Cathepsin G to Gonococci
More LessHuman polymorphonuclear leucocyte (PMN) lysosomal cathepsin G exerts potent bactericidal action against Neisseria gonorrhoeae in vitro, independent of its serine esterase activity. The results presented demonstrate that (1) bactericidal, diisopropylfluorophosphate-treated cathepsin G binds in a specific and saturable manner to the surface of gonococci, (2) loss of carbohydrates in gonococcal LPS due to mutation increases total and specific binding of cathepsin G, and (3) at least three outer-membrane proteins (OMPs) (PIA, PIII. and a 45 kDa OMP) interact with cathepsin G. Taken together, the results suggest that gonococcal susceptibility to the lethal action of cathepsin G, and perhaps susceptibility of gonococci to oxygen-independent killing by PMNs, is controlled by LPS-masking of cathepsin-G-binding OMPs.
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DNA Hybridization with Hardjobovis-specific Recombinant Probes as a Method for Type Discrimination of Leptospira interrogans Serovar hardjo
More LessRestriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.
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Nucleotide Sequence of the Pilin Gene of Bacteroides nodosus 340 (Serogroup D) and Implications for the Relatedness of Serogroups
More LessThe gene encoding pilin of Bacteroides nodosus 340 has been isolated and the nucleotide sequence determined. The gene is present as a single copy within the B. nodosus genome and a protein of M r 16683 can be predicted from the proposed coding region. A comparison of the predicted amino acid sequence with pilin from other strains of B. nodosus indicated that the protein of strain 340 (serogroup D) has a high degree of similarity with pilin of strain 265 (serogroup H). The degree of similarity between pilins from these strains and from other B. nodosus serogroups is no greater than that between B. nodosus pilins and the homologous proteins of several different bacterial species. These findings suggest that serogroups D and H may form a subset of B. nodosus serogroups.
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Effect of Dilution Rate and Mg2+ Limitation on Toxic Shock Syndrome Toxin-1 Production by Staphylococcus aureus Grown in Defined Continuous Culture
More LessA toxic shock syndrome isolate of Staphylococcus aureus was grown in a chemostat, in a defined synthetic medium of six amino acids, glucose, two vitamins and salts. Steady states were achieved under limiting and replete Mg2+ conditions and at a range of relative specific growth rates. The biomass and toxic shock syndrome toxin-1 (TSST-1) were estimated at each condition. Under Mg2- limitation the biomass and TSST-1 production rates were reduced compared to Mg2+ replete conditions. Optimal TSST-1 production occurred at 0.81 relative specific growth rate.
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Vero Cytotoxin Production and Presence of VT Genes in Escherichia coli Strains of Animal Origin
More LessVero cytotoxin (VT) production has been studied in 34 Escherichia coli strains isolated from animals with enteric diseases. The strains were tested by DNA hybridization with probes for VT1 and VT2 sequences and also in toxin neutralization experiments with specific antisera. Twenty bovine strains, belonging to nine different O serogroups, produced VT1 or VT2 but not both toxins. In contrast, all 14 porcine strains of four different O serogroups produced VT2 only. Six of these porcine strains, belonging to serogroups 0138, 0139 and 0141, were isolated from cases of oedema disease. In general, the porcine isolates produced toxin at a lower level than the bovine strains.
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Molecular Cloning of the Shv-1 β-Lactamase Gene and Construction of an Shv-1 Hybridization Probe
More LessWe have cloned the Shv-1 β-lactamase gene from the R1010 plasmid into pACYC184. By subcloning and transposon mutagenesis we have localized the gene to a 1·6 kb BscI-SalI fragment of R1010, which is present in the recombinant plasmid pUB8. A 900 bp PstI fragment of pUB8 was shown to be a specific hybridization probe by testing against plasmids which encode 17 different β-lactamase enzymes. A comparison was made of the sensitivity of the Shv-1 probe labelled with either [35S]dCTP or with photobiotin.
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- Physiology And Growth
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The Role of Glycerol in Osmotolerance of the Yeast Debaryomyces hansenii
More LessTransfer of growing cells of the salt-tolerant yeast Debaryomyces hansenii to media of higher salinity resulted in an increased production and intracellular accumulation of glycerol, which was proportional to the magnitude of the shift in salinity. Stress solutes other than NaCl, when added in iso-osmolar concentrations, promoted the accumulation of similar amounts of glycerol. Cells grown at high salinity rapidly lost glycerol when returned to media of lower salinity and the loss was greater when the cells were transferred to more dilute media. A mutant strain of D. hansenii showed poor glycerol production and was inhibited by NaCl at concentrations about half the maximum tolerated by the wild-type. Growth of this mutant occurred at otherwise inhibitory NaCl concentrations if the medium was supplemented with a low concentration of glycerol. The added glycerol was intracellularly accumulated to levels that increased with salinity and were only slightly lower than the corresponding wild-type levels. Glycerol additions above the growth promoting level had little effect on growth rate but caused substantial shortening of the lag phase. Osmoprotectants other than glycerol did not permit growth to occur. The mutant was isolated as a glycerol non-utilizer but displayed growth in glycerol media at increased NaCl concentrations.
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Extraordinarily Rapid Appearance of a β-Fructofuranosidase (Exo-inulase)-hyperproducing Mutant in Continuous Culture of Kluyveromyces fragilis
More LessA wild-type strain of the yeast Kluyveromyces fragilis was grown in continuous culture on inulin-, fructose- and glucose-limited media. In the presence of organic nitrogen, all three carbon sources supported an extremely rapid changeover to a homogeneous mutant cell population that exhibited hyperproduction of β-fructofuranosidase (exo-inulase). The non-specific role of the carbon substrate in the takeover suggested co-regulation of inulase synthesis and early glycolytic pathway enzymes such as hexokinase, resulting in gratuitous hyperproduction of inulase. The rate of mutant appearance increased linearly with a decrease in dilution rate as well as carbon/nitrogen ratio, implying that the responsible forces were related to the residual concentration of the limiting carbon substrate. Under optimum conditions, mutant cells were detected within only 40 h (2·9 generations) from the start of continuous culture, at least 6·5 times faster than predicted. The maximum rate of displacement was as high as 54% per generation, or more than 12 times greater than predicted from the differential growth rate between the wild-type and mutant strain. These extraordinarily rapid takeovers point to a very fast and ongoing genetic change triggered by growth under severe stress.
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Mycobactins and Exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and Other Related Species
More LessFollowing iron-deficient growth, mycobactins and exochelins were isolated from 11 strains of Mycobacterium tuberculosis (including type strains of the virulent H37Rv and avirulent H37Ra organisms) as well as from M. bovis (one strain), M. bovis BCG (two strains), M. africanum (eight strains) and M. xenopi (one strain) but not from M. microti (one strain). The mycobactins from the tuberculosis group (M. tuberculosis, M. bovis and M. africanum) were identical and could each be resolved into four compounds by thin-layer chromatography (TLC) and into several fractions by high-performance liquid chromatography, supporting previous claims that this group is a single taxon. Exochelins were all chloroform-soluble and showed no species specificity; no single exochelin was recognized by TLC that had not been previously seen in M. avium or a related species.
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Continued Growth of Escherichia coli after Stopping Medium Addition to a K+-limited Chemostat Culture
More LessThe steady-state bacterial dry wt of Escherichia coli, growing under K+-limited conditions in the chemostat, was inversely dependent on the growth rate. This phenomenon was more carefully investigated in medium-flow stop experiments. Growth did not stop immediately but continued for a time, initially at the same rate as before. The dry wt increased to a value corresponding to a steady-state growth rate near zero, independent of the initial specific growth rate. This was observed in both the wild-type strain and a mutant that lacked the high-affinity K+ uptake system. The wild-type strain maintained a low extracellular K+ concentration both in the chemostat under steady-state conditions and after stopping the medium flow. The mutant, on the other hand, maintained a much higher extracellular K+ concentration in the steady state, which decreased to much lower values after stopping the medium flow. From the increase in bacterial dry wt and the low external K+ concentration after stopping the medium flow it is concluded that the intracellular K+ is redistributed among the cells, including new cells. The growth yield on K+ was highest in the stationary growth phase of a batch culture and all steady-state cultures converged ultimately to this yield value after the medium flow had been stopped. It is proposed that the growth rate of E. coli under K+-limited conditions is determined by the intracellular K+ concentration.
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Regulation of Trehalase Activity during the Cell Cycle of Saccharomyces cerevisiae
More LessSynchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle. After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures. Both synchronous and control cultures were studied for two cell cycles. In asynchronous cultures the trehalase activity of crude cell lysates rose continuously. In synchronized populations trehalase activity increased from the beginning of budding onwards. However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold. The same changes were found during the second budding cycle. Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes. Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures. In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity. These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism.
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Presence of Anaplerotic Reactions and Transamination, and the Absence of the Tricarboxylic Acid Cycle in Mollicutes
More LessCell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.
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An Extracellular Blood-anticoagulant Glycopeptide Produced Exclusively during Vegetative Growth by Myxococcus xanthus and Other Myxobacteria Is Not Co-regulated with Other Extracellular Macromolecules
More LessWe have shown that the blood anticoagulant activity (BAA) secreted by Myxococcus xanthus (designated myxaline) is a heat-stable molecule: a high-molecular-mass extracellular fraction also with an apparent BAA is a thermolabile protease. This property allowed us to assay the BAA content in crude boiled culture supernatants and to study the conditions under which it is produced. Heat-stable BAA is strictly extracellular and its production is restricted to vegetative growth in M. xanthus. Unlike the other extracellular proteins, its production is not affected by mutations that regulate secretion; mutations that modify the extracellular proteolytic activity do not modulate the amount of myxaline produced either. Several other species of Myxococcus and one other myxobacterial species produce a heat-stable BAA during vegetative growth.
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- Systematics
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Taxonomic Study of Non-alkaliphilic Halococci
More LessNinety-six extremely halophilic, non-alkaliphilic cocci were isolated from several salterns in different geographical areas of Spain. These strains, together with seven reference strains of the genus Halococcus, were characterized by means of 114 phenotypic features, the results being analysed by numerical techniques using the simple matching (SSM ) coefficient and the unweighted pair group clustering (UPGMA) algorithm. At the 70% similarity level, four phenons were obtained. Phenon A contained 87 strains, including all the reference strains, and was considered to comprise members of the only named species of the genus Halococcus, H. morrhuae. Phenons B and C, which included five and seven strains respectively, showed greater metabolic versatility than phenon A. The four strains belonging to phenon D were significantly different from the other phenons in that they produced acid from glucose and were able to use most of the organic compounds tested. The results indicate that there is phenotypic diversity among the members of the genus Halococcus and that phenon D may constitute a new taxon.
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Numerical Taxonomy of Moderately Halophilic Gram-negative Rods from an Inland Saltern
A total of 174 moderately halophilic bacteria was isolated from an inland saltern with athalassohaline characteristics, located in La Malá, Granada, Spain. The results of 118 phenotypic tests were submitted to a numerical analysis with ten reference strains using simple matching (SSM ) and Jaccard (SJ ) coefficients. Clustering was done by the unweighted pair-group method of association (UPGMA), single linkage and complete linkage analysis. Five phenons were found at the 67% similarity level (SSM ). Phenon A included 74 strains assigned to Vibrio; 22 bacteria belonging to phenon B were assigned to the genus Alteromonas; strains of phenon C were assigned to Deleya (43 strains) and Acinetobacter (7 strains); phenon D resembled Pseudomonas (13 strains); phenon E could be related to Flavobacterium oceanosedimentum (9 strains).
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An Assessment of Taxonomic Congruence between DNA—DNA Hybridization and Pyrolysis Gas—Liquid Chromatographic Classifications
More LessThe existence of subgroups within Bacillus megaterium has been reported previously on the basis of DNA-DNA hybridization and DNA base composition studies. In this study the strains used to define these subgroups have been reanalysed by pyrolysis gas-liquid chromatography. The resultant two-group classification of the test strains was directly comparable with that obtained from the previous nucleic acid analyses at the between-group level. However, comparisons of the test strains at the within-group level proved less successful.
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Biochemical and Physiological Characterization of Colony Form Variants in Xenorhabdus spp. (Enterobacteriaceae)
More LessPrimary and secondary form variants of Xenorhabdus isolated from 21 strains (13 species) of Steinernematidae and Heterorhabditidae were tested for 240 biochemical and physiological characters. Primary form variants, isolated from the infective stage nematodes, could always be distinguished from the secondary by adsorption of neutral red from MacConkey agar. Lecithinase, antibiotic activity and/or adsorption of bromothymol blue were useful for distinguishing the variants of most strains. The variants of all strains also differed for other characteristics but the distinguishing characteristics varied from strain to strain. The importance of including both variants of each strain and of using appropriate methods in the study of Xenorhabdus taxonomy was demonstrated.
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