- Volume 134, Issue 7, 1988
Volume 134, Issue 7, 1988
- Biochemistry
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Thermostable Peroxidase from Bacillus stearothermophilus
More LessA peroxidase from Bacillus stearothermophilus was purified to homogeneity. The enzyme (M r 175000) was composed of two subunits of equal size, and showed a Soret band at 406 nm. On reduction with sodium dithionite, absorption at 434 nm and 558 nm was observed. The spectrum of reduced pyridine haemochrome showed peaks at 418, 526 and 557 nm; the reduced minus oxidized spectrum of pyridine haemochrome showed peaks of 418, 524 and 556 nm with a trough at 452 nm. These results indicate that the enzyme contained protohaem IX as a prosthetic group. The optimum pH was about 6 and the apparent optimum temperature was 70 °C. The enzyme was relatively stable up to 70 °C; at 30 °C it was stable for a month. The enzyme had peroxidase activity toward a mixture of 2,4-dichlorophenol and 4-aminoantipyrine with a K m for H2O2 of 1·3 mm. It also acted as a catalase with a K m for H2O2 of 7·5 mm.
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Mechanism of Gliotoxin Action and Factors Mediating Gliotoxin Sensitivity
More LessResistance to the low M r fungal epipolythiodiketopiperazine toxin, gliotoxin, varied threefold between the phytopathogens Pythium ultimum and Rhizoctonia solani. Uptake of radiolabelled gliotoxin was rapid and concentration dependent. Uptake by P. ultimum was largely complete within 1 min while the rate of uptake peaked within 10 min for R. solani (anastomosis groups 2-2 and 4). Uptake of gliotoxin by P. ultimum was twice that shown by the more resistant R. solani. A deep rough mutant of Salmonella typhimurium, deficient in outer-membrane polysaccharide synthesis, was hypersensitive to gliotoxin, indicating that diffusion barriers play a role in relative sensitivity to gliotoxin. Fungal glutathione levels (reduced and oxidized) did not differ appreciably before or after gliotoxin exposure, indicating that this cytoplasm-based detoxification mechanism was not important in the relative fungal sensitivity to gliotoxin. Binding of the radiolabelled thiol reagents N-ethylmaleimide (NEM) and iodoacetic acid to fungal thiol groups was inhibited by gliotoxin. Conversely, the thiol reagents NEM and p-chloromercuribenzoic acid inhibited the uptake of radiolabelled gliotoxin. Uptake of radiolabelled amino acids and glucose was reduced by up to 85% by gliotoxin (8 μg ml−1). It is suggested that the primary mechanism of action of gliotoxin involves selective binding to cytoplasmic membrane thiol groups.
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- Development And Structure
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Studies on Cell Division in Regenerating Protoplasts of the Yeast Schizosaccharomyces japonicus
More LessRegeneration of the cell wall in yeast protoplasts can be blocked by the action of snail enzymes. Electron microscopic studies of protoplasts of Schizosaccharomyces japonicus var. versatilis showed that incubation in the presence of snail enzymes resulted in the production of incomplete cell walls consisting of α-(1 → 3)-glucan microfibrils aggregated into flat sheets of irregular size. This incomplete cell wall did not allow the formation of a septum and subsequent cell division. In contrast, protoplasts of the same yeast species growing in a flattened state produced by physical constraint formed incomplete walls that permitted cytokinesis but not reversion of the protoplasts to normal cells. These incomplete walls comprised three structural components: (i) a continuous network of long β-(1 →3)-glucan microfibrils; (ii) short α-(1 →3)-glucan microfibrils; and (iii) an amorphous matrix. This ultrastructural picture corresponded to the first stages of regeneration (reached after 2–3 h incubation) leading to a complete cell wall; the walls in the flattened protoplasts, however, were not completed and consequently the protoplasts did not revert to normal cells.
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- Ecology
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Adsorption of the Rhizosphere Bacterium Azospirillum brasilense Cd to Soil, Sand and Peat Particles
More LessThe rhizosphere bacterium Azospirillum brasilense Cd adsorbed strongly to light-texture and heavy-texture soils, but only slightly to quartz sand. Increase in clay and organic matter content, decrease in soil pH, or flooding the soil enhanced adsorption, whereas the presence of a bacterial attractant, increase in soil pH or drying of the soil decreased adsorption. The cells adsorbed to the upper fraction of the soil profile, but were able to infiltrate deeper if very dry soil was wetted. Washing the soil did not desorb the bacteria from soil particles, but it did from the sand particles. Overwashing soil recovered relatively few cells, whereas overwashing sand detached most of the bacteria. Survival time of A. brasilense Cd was short in soil but long in peat inoculant.
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- Genetics And Molecular Biology
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Characterization of Bacteriophage ϕC69 of Saccharopolyspora erythraea and Demonstration of Heterologous Actinophage Propagation by Transfection of Streptomyces and Saccharopolyspora
More LessA bacteriophage, designated ϕC69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. ϕC69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with ϕC69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.
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Characterization of SE-3, a Virulent Bacteriophage of Saccharopolyspora erythraea
More LessSE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer. The host range of the phage is narrow, limited to some strains of this species. Another strain of Sac. erythraea, and a strain of Sac. hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication. SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping. The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.
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Construction of Multicopy Expression Vectors for Regulated Over-production of Proteins in Klebsiella pneumoniae and Other Enteric Bacteria
More LessA number of expression vectors have been constructed to allow over-production of selected gene products in Klebsiella pneumoniae and other enteric bacteria. The plasmids use the strong hybrid trp-lac (tac) promoter for gene expression, which is regulated by the lacI Q allele of the lac repressor carried on the vector. This provides very tight regulation of gene expression, which is important for over-production of proteins which may be detrimental to cell growth. The vectors carry the standard mpl8 cloning nest in which all the restriction sites are unique to the plasmid (with the exception of EcoRI in pDK7). Derivatives were constructed carrying kanamycin, chloramphenicol or ampillicin resistance as selectable markers, the first two of which are advantageous in K. pneumoniae due to the high inherent -lactamase activity of this organism.
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Segmented Arrangement of Borrelia duttonii DNA and Location of Variant Surface Antigen Genes
More LessThe DNA of an isolate of Borrelia duttonii, an agent of relapsing fever is present as seven major species ranging in size from 10 kb to greater than 150 kb. Additionally, this isolate contains low copy number species, both smaller and larger than these seven major elements. No one of these individual DNA species obviously corresponds to the bacterial chromosome, unlike the situation in Borrelia hermsii, another relapsing fever Borrelia. Thus it appears that B. duttonii has a unique segmented arrangement of its genetic material. Cloned DNA fragments containing coding sequences specific for variant surface antigens of B. duttonii hybridize to a closely migrating, high copy number subset of these genetic elements.
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Cloning and Nucleotide Sequence of the Highly Thermostable Neutral Protease Gene from Bacillus stearothermophilus
More LessThe gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promoter sequence (TTTTCC for the –35 region and TATTGT for the – 10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upsteam of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.
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Molecular Cloning and Characterization of Bacillus alvei Thiol-dependent Cytolytic Toxin Expressed in Escherichia coli
More LessA chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (M r 60000, pI 5·0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64kDa polypeptide of pI 8·2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.
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Mixed-Ligand Complexes of Platinum(II) as Curing Agents for pBR322 and pBR329 (ColE1) Plasmids in Escherichia coli
More LessThe mixed-ligand complexes of platinum(II) [Pt(Gly)(uracil)Cl2].H2O and [Pt(His)(adenine)]-Cl.2H2O eliminated multicopy plasmids with the ColE1 origin of replication in Escherichia coli with 100% frequency. However, plasmids of the IncF1, H1 and X groups were totally refractory to these agents under similar conditions.
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Characterization of Two Families of Spontaneously Amplifiable Units of DNA in Streptomyces ambofaciens
More LessFour highly amplified DNA sequences (ADS) ranging from 5·8 to 24·8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24·8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.
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Characteristics of RP4 Tellurite-resistance Transposon Tn521
More LessA restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).
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DNA-mediated Transformation in the Aquatic Filamentous Fungus Achlya ambisexualis
More LessA DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neoμm. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the trans-formant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.
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Loss of the Toluene-Xylene Catabolic Genes of TOL Plasmid pWWO during Growth of Pseudomonas putida on Benzoate Is Due to a Selective Growth Advantage of ‘Cured’ Segregants
More LessDuring growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative (‘cured’) strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWWO. Experiments with two plasmids identical to pWWO but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the ‘benzoate caring’ occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWWO-174, a derivative of pWWO which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWWO, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the ‘cured’ phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously ‘cured’ derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10−2 and 10−3. We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the ‘cured’ segregants grow faster than the wild-type using the chromosomally determined β-ketoadipate pathway.
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- Pathogenicity And Medical Microbiology
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Serotype-specific Monoclonal Antibodies against the H12 Flagellar Antigen of Escherichia coli
The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament. Reexamination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology. Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the M r of individual flagellins. There was no apparent correlation between these two features. Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella. In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different 0:K antigen combinations. The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains. The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers. The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament. The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.
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Stability of Plasmid Sequences in an Acute Q-Fever Strain of Coxiella burnetii
More LessThe rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host. Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors. Genetically, the regions of DNA responsible for phase variation have not been identified. We have sought to determine whether the plasmid identified in acute disease isolates, QpHl, which represents approximately 5% of the coding capacity of this organism is involved in phase variation. Plasmids from phase 1 and phase 2 variants (designated QpHl and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells. Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage. The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type. Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E. coli extract was also identical. Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical. These data indicate that within the limits of our analysis, the plasmid DNA from C. burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation.
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Characterization of Two Haemophilus somnus Fc Receptors
More LessHaemophilus somnus expresses two types of receptors that bind to the Fc region of bovine IgG, IgA and IgM. In this study, the relationship between these two types of Fc receptors is characterized. The high molecular mass receptors (350, 270 and 120 kDa) were secreted into the culture medium and were also in the insoluble protein fraction of the culture medium. The 41 kDa Fc receptor, which is a major outer-membrane protein, was only present in the insoluble protein fraction. Peptide mapping of the two types of Fc receptors suggests that the 41 kDa receptor is related to the high molecular mass receptor complex. Disulphide linkage is unlikely to be the mechanism of association of the 41 kDa receptor with the high molecular mass receptors since reducing agents had no effect on separating the individual receptors. Although the 41 kDa receptor is a major protein in the outer membrane of H. somnus, it does not react with convalescent bovine sera in Western blots. In contrast, convalescent bovine sera reacts intensely with the high molecular mass receptors in Western blots.
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Differences among Shigella spp. in Susceptibility to the Bactericidal Activity of Human Serum
Clinical isolates of Shigella spp. were examined for their susceptibility to human serum. The susceptibility of the strains to immune and nonimmune human serum was dependent upon the size of the bacterial inoculum and the concentration of serum. There were differences among Shigella spp. in susceptibility to human serum: S. sonnei strains were the least susceptible, strains of S. boydii and S. flexneri serotype 6 were intermediate, and those of S. flexneri other than serotype 6 and S. dysenteriae were the most susceptible. Experiments in which heat-treated (56 °C for 30 min, or 50 °C for 20 min) serum was used, and analysis of activation of complement by lipopolysaccharides (LPS) from each Shigella sp., suggested that LPS composition, especially the O antigen polysaccharide chains, contributes to the differences among Shigella spp. in susceptibility to human serum.
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An in vitro Model of Chlamydia trachomatis Infection in the Regenerative Phase of the Human Endometrial Cycle
More LessAn in vitro model of the regenerative phase of the human endometrial cycle was developed in order to study the growth of Chlamydia trachomatis during the period following menses. Glandular epithelial fragments were prepared from curettings of endometria and explanted onto coated substrata. Epithelial cells migrated rapidly from the explant in a fashion which closely mimicked the regeneration of the surface epithelium after menses. The cultures were then experimentally infected with C. trachomatis serotype E at various times during formation of the outgrowth. Chlamydial inclusions developed both within the explants and in the outgrowing epithelial sheets. They were also found in isolated epithelial and non-epithelial cells. However, the most striking feature of chlamydial inclusion development within these cultures was the tendency for inclusions to be located in cells at the periphery of the epithelial sheets. This was partly due to the failure of the cells within the sheets to bind chlamydiae after centrifugation of the organisms onto the culture and partly due to a phenomenon similar to phagokinesis. During this process infectious chlamydial particles were cleared from the substratum by migrating cells with free motile edges, which occasionally led to internalization and inclusion development within these cells.
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Plasmid-mediated Serum Resistance and Alterations in the Composition of Lipopolysaccharides in Salmonella dublin
More LessSurvival rates of Salmonella dublin in rabbit serum after culture for 1 h at 37 °C were compared between a wild-type strain (5240) carrying a 50 MDa plasmid, a plasmid-cured strain (C524), and a cured strain containing the 50 MDa plasmid tagged with TnI (5241). Strain C524 was more susceptible to the bactericidal activity of normal serum than its parent strain 5240 (percentage survival < 1 % and 52·5 ± 9·2%, respectively). On the other hand, the percentage survival of strain 5241 was significantly increased (90·4 ± 4·0%), indicating that the reintroduction of the plasmid into the cured strain restored the serum resistance. Moreover, this change in the serum resistance properties correlated with changes in the neutral sugar composition of the lipopolysaccharides (LPS) of these strains, suggesting that the 50 MDa plasmid is necessary for O-side chain expression in the LPS of S. dublin.
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- Physiology And Growth
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Aerobic Nitrogen Fixation in Aggregate-forming Cultures of the Nonheterocystous Cyanobacterium Microcoleus chthonoplastes
More LessMicrocoleus chthonoplastes is a filamentous, nonheterocystous cyanobacterium which fixes gaseous nitrogen in axenic culture under aerobic conditions. Growth on agar plates reflects the benthic habit observed in the natural environment, and in liquid culture clumps or aggregates are produced. Scanning electron microscopy suggests that these aggregates are primarily the result of random movement of trichomes against one another. The aim of this study was to ascertain whether aggregation or any intracellular differentiation might be important in protection of the oxygen-labile nitrogenase in this species. Observations using light and transmission electron microscopy revealed no cellular or intracellular differentiation, and tetrazolium labelling experiments provided no evidence that a proportion of cells might act in a heterocyst-like fashion. The results presented suggest that nitrogen fixation in M. chthonoplastes is unlikely to be enhanced in oxygen-deficient microsites formed as a result of aggregation.
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Fixation of N2 by Bacteroids from Stem Nodules of Sesbania rostrata
More LessBacteroids, prepared from stem nodules of Sesbania rostrata inoculated with Rhizobium sp. strain ORS571, in steady-state reactions in which O2 was supplied in solution with soybean leghaemoglobin or mammalian myoglobin as O2 carriers, and with succinate as exogenous substrate, fixed N2 to NH3 with highest rates at 10 nM free O2, where O2 uptake was approximately half-maximal. Higher concentrations (> 100 nM) of free O2, shown previously to be required for optimum nitrogenase activity in O2-trained ORS571 grown in continuous culture, inhibited N2 fixation by stem nodule bacteroids.
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Metabolic Changes during Development of Phytophthora palmivora Examined by Gas Chromatography/Mass Spectrometry
More LessMetabolic profiles from four stages of differentiation of the fungus Phytophthora palmivora were obtained by gas chromatography/mass spectrometry. The profiles showed the presence of sterols in the asexual reproduction stage of the organism, and confirmed their virtual absence from the mycelial stages. The zoospore stage was characterized by the presence of polyunsaturated fatty acids of C20 and C22 chain length. The transition from zoospore to cyst was also marked by the appearance of disaccharides and by a decrease in the amount of phosphate present. There were also distinctive shifts in the proportions and the total amounts of amino acids present, with γ-aminobutyrate and alanine increasing as germination took place. These distinctive profiles identify some of the metabolic changes which accompany differentiation in this fungus.
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Efficiency and Stability of Exopolysaccharide Production from Different Carbon Sources by Erwinia herbicola
More LessUnder conditions of glucose limitation the maximum yield of Erwinia herbicola from oxygen (Y max o2 ) and glucose (Y max gic) was 31 g mol−1 and 72 g mol−1 respectively. This corresponded to a relative ATP/O quotient between 1.1 and 1.5. The cytochrome profile indicated the absence of c-type cytochromes which is consistent with this low ATP/O quotient. During nitrogen-limited growth on glucose, the rate of polysaccharide production (0·34-0·37 g g−1 h−1) was independent of growth rate, whereas the rate of production of gluconate and 2-ketogluconate increased with growth rate. The ATP/O quotient of 1.25 derived from glucose-limited growth was used to calculate the theoretical yields of exopolysaccharide from fructose and oxygen. The values obtained were close to the observed yields once an allowance had been made for cell production. Thus the growth efficiency of E. herbicola appears to be unaltered during carbon- or nitrogen-limited growth on fructose. At an ATP/O quotient of 1.25 the synthesis of the acid moiety of the exopolysaccharide provides a significant proportion (51–65%) of the ATP needed for polymerization of the sugar backbone. Nevertheless, the rate of ATP turnover is large in relation to that required for cell production under these conditions. Polymer production may serve as a means of turning over ATP but it is not essential for growth as mutants that do not produce exopolysaccharide but turnover ATP by some other means are readily selected. The rate of ATP turnover rather than exopolysaccharide production appears to be physiologically important.
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Effect of Hexadecane-induced Vesiculation on the Outer Membrane of Acinetobacter calcoaceticus
More LessLipopolysaccharide-rich vesicles were released from Acinetobacter calcoaceticus 69V during growth on hexadecane. Vesicle formation occurred over the whole surface of the cell as demonstrated by scanning electron microscopy. In contrast, the surface of acetate-grown cells, for which little lipopolysaccharide was found in the growth medium, appeared smooth. The overall chemical composition as well as the protein and phospholipid composition of the outer membranes of both cell types was very similar. In the vesicles all outer membrane proteins were found with the exception of an M r 10000 polypeptide corresponding to Braun’s lipoprotein. Compared with the outer membrane, the vesicles contained more phosphatidylethanolamine. Hexadecane-grown cells were susceptible to exogenously added phospholipase. Nevertheless the barrier function towards lysozyme was retained.
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- Systematics
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Nitrogen Fixation by Marine Agar-degrading Bacteria
More LessSeveral strains of agar-degrading bacteria capable of fixing N2 were isolated from seawater and eelgrass-bed sediment in Aburatsubo Inlet, Kanagawa, Japan, during the summer of 1986. All strains were Gram-negative, facultatively anaerobic, and required NaCl for growth. They were straight or slightly curved rods and were motile in liquid medium by means of a single polar flagellum. These characteristics as well as the G+C contents of their DNA (44·7–46·1 mol%) placed them in the family Vibrionaceae. These strains produced extracellular agarase on agar medium, yielding reducing sugars and acids as the end products. They expressed significant nitrogenase (acetylene reduction) activities after a few hours of incubation under anaerobic conditions. They utilized combined nitrogen sources both aerobically and anaerobically, but fixed N2 only under anaerobic conditions. Neither yeast extract nor vitamins were required for N2 fixation. These strains were demonstrated to fix N2 anaerobically using agar as the sole carbon source.
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O-Antigenic Lipopolysaccharides Isolated from a Marine Vibrio, Bio-serogroup 1875, Possessing an Antigenic Factor in Common with O1 Vibrio cholerae
More LessThe chemical and serological characteristics of lipopolysaccharides (LPS) isolated from Vibrio bio-serogroup 1875 were compared with those of O1 Vibrio cholerae LPS. Vibrio bio-serogroup 1875 LPS contained all the component sugars which were found in O1 V. cholerae LPS, i.e. glucose, l-glycero-d-manno-heptose, fructose, glucosamine, perosamine and quinovosamine, though the amount of perosamine, a characteristic component of O1 V. cholerae LPS, was very low compared with that of O1 V. cholerae LPS. Their LPS additionally contained mannose and two unidentified neutral sugars which are not regular constituents of O1 V. cholerae LPS. Definite serological cross-reactivity in the passive haemolysis test between LPS from Vibrio bio-serogroup 1875 and LPS from O1 V. cholerae was demonstrated.
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A Numerical Taxonomic Study of the Genus Xenorhabdus (Enterobacteriaceae) and Proposed Elevation of the Subspecies of X. nematophilus to Species
More LessData from a study of both phases of 21 strains of Xenorhabdus examined for 240 characters were subjected to numerical analysis. Only 60 characters were used for the analyses, since 169 characters were common to all isolates, and the acidification data essentially duplicated the assimilation tests. The data were arranged in seven ways to determine the significance of characters affected by phase change and of weak responses. Most of the analyses involved calculation of similarities by the Jaccard coefficient and clustering by single linkage, complete linkage and centroid sorting algorithms. The resultant dendrograms emphasized the importance of recognizing phase-related characteristics in examining the taxonomy of Xenorhabdus. They also demonstrated a close correspondence between the taxonomic groupings of Xenorhabdus and those of their nematode associates. It is proposed that the subspecies of X. nematophilus be elevated to species, X. nematophilus, X. bovienii, X. poinarii and X. beddingii.
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A Numerical Classification of the Genus Bacillus
More LessThree hundred and sixty-eight strains of aerobic, endospore-forming bacteria which included type and reference cultures of Bacillus and environmental isolates were studied. Overall similarities of these strains for 118 unit characters were determined by the SSM, SJ and DP coefficients and clustering achieved using the UPGMA algorithm. Test error was within acceptable limits. Six cluster-groups were defined at 70% SSM, which corresponded to 69% SP and 48-57% SJ. Groupings obtained with the three coefficients were generally similar but there were some changes in the definition and membership of cluster-groups and clusters, particularly with the SJ coefficient.
The Bacillus strains were distributed among 31 major (4 or more strains), 18 minor (2 or 3 strains) and 30 single-member clusters at the 83% SSM level. Most of these clusters can be regarded as taxospecies. The heterogeneity of several species, including Bacillus brevis, B. circulans, B. coagulans, B. megateriun, B. sphaericus and B. stearothermophilus, has been indicated and the species status of several taxa of hitherto uncertain validity confirmed. Thus on the basis of the numerical phenetic and appropriate (published) molecular genetic data, it is proposed that the following names be recognized; Bacillus flexus (Batchelor) nom. rev., Bacillus fusiformis (Smith et al.) comb. nov., Bacillus kaustophilus (Prickett) nom. rev., Bacillus psychrosaccharolyticus (Larkin & Stokes) nom. rev. and Bacillus simplex (Gottheil) nom. rev. Other phenetically well-defined taxospecies included “B. aneurinolyticus”, “B. apiarius”, “B. cascainensis”, “B. thiaminolyticus” and three clusters of environmental isolates related to B. firmus and previously described as “B. firmus-B. lentus intermediates”. Future developments in the light of the numerical phenetic data are discussed.
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The Significance of Long-chain Fatty Acid Composition and Other Phenotypic Characteristics in Determining Relationships Among Some Pichia and Candida Species
More LessThe long-chain fatty acid compositions of 22 species of Candida were determined, and compared with the fatty acid compositions of 10 species of the genus Pichia that contain coenzyme Q9. The long-chain fatty acid results were also compared with other phenotypic criteria (i.e. assimilation of carbon sources, coenzyme Q type, G + C content and proton magnetic resonance spectra) in order to establish possible anamorph/teleomorph relations. Close correlations were found between known perfect/imperfect states. The results suggest that C. cacaoi and P.farinosa, and C. maltosa and P. etchellsii, also have anamorph/teleomorph relationships.
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Comparison of 16S rRNA Sequences from the Family Pasteurellaceae: Phylogenetic Relatedness by Cluster Analysis
More LessThe taxonomy of the family Pasteurellaceae has remained controversial despite investigations of biochemistry, serology, and nucleic acid relatedness. In an attempt to resolve some of this confusion, we have partially sequenced the 16S rRNAs of seven members of the family, representing all three genera. The sequences were aligned, similarity scores calculated, and single, average and complete linkage cluster analysis of the resulting distance matrix performed. In this way, an evolutionary branching pattern of these closely related species was recontructed, and the approximate phylogenetic position of the family determined. Actinobacillus (Haemophilus) actinomycetemcomitans clustered with Haemophilus instead of Actinobacillus, supporting transfer of this species to the genus Haemophilus. Thus cluster analysis of phylogenetic relatedness was found to be particularly useful for studying closely related organisms, and could be performed using a microcomputer.
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Synthetic Oligodeoxynucleotide Probes for the Rapid Detection of Bacteria Associated with Human Periodontitis
More LessAnalysis of the subgingival microflora has recently implicated Actinobacillus (Haemophilus) actinomycetemcomitans and several black Bacteroides species in the aetiology of juvenile, adult and rapidly progressing periodontitis. Rapid bacteriological diagnosis has been hampered by the slow growth and fastidious nature of these bacteria. To construct diagnostic probes, dideoxy sequencing of the 16S rRNA molecules from A. (H.) actinomycetemcomitans, Haemophilus aphrophilus, Bacteroides gingivalis, Bacteroides intermedius subgroup II, Bacteroides asaccharolyticus and several closely related species was performed. Next, oligodeoxynucleotides, complementary to defined regions of the 16S rRNA exhibiting considerable evolutionary divergence, were synthesized for use as molecular probes. In a dot-blot hybridization assay, all strains from each of the species for which probes were constructed were correctly identified, with a detection limit of less than 5 103 organisms. No cross-hybridization to closely related species (except for H. aphrophilus and Haemophilus paraphrophilus) or contaminating bacteria was observed. Using a modified DNA/RNA hybridization technique, the detection could be performed in less than 12 h, as compared to 2-3 weeks using conventional bacteriological procedures.
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Phenotypic Diversity in Pseudomonas syringae pv. tomato
More LessTwenty-nine strains of Pseudomonas syringae pv. tomato (P. s. tomato) that represented the temporal and geographical diversity of this pathogen were tested for pathogenicity on tomato, carbohydrate utilization, bacteriophage sensitivity, fatty acid composition and plasmid profile. The extent of phenotypic diversity observed within P. s. tomato depended on the trait examined; the strains were similar in pathogenicity, carbohydrate utilization and fatty acid content, whereas greater diversity was found in bacteriophage sensitivity and the plasmid profiles. A classification scheme for P. s. tomato plasmids based on both size and DNA homology is proposed. The array of phenotypic traits clearly differentiated all the strains of P. s. tomato examined from six strains of P. syringae pv. syringae, with carbohydrate utilization and fatty acid analyses being the most reliable.
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Genetic Diversity and Relationships of Two Pathovars of Pseudomonas syringae
More LessTo determine genetic relationships within and between two pathovars of Pseudomonas syringae, strains typical of P. syringae pv. tomato (P. s. tomato) and selected strains of P. syringae pv. syringae (P. s. syringae) were characterized by three methods. DNA-DNA hybridization experiments showed that strains of P. s. tomato and P. s. syringae were, respectively, 86-100% and 37–47% homologous to DNA from a P. s. tomato reference strain when tested under stringent conditions. An analysis of electrophoretic variation in enzymes encoded by 26 loci placed 17 P. s. tomato strains studied in a group of four electrophoretic types, and these strains had a mean genetic diversity per locus of 0·076. Six P. s. syringae strains formed a second group of six electrophoretic types, which had a higher mean genetic diversity per locus of 0·479. The mean genetic distance separating P. s. tomato from P. s. syringae (D = 0·94) was unexpectedly large for strains of a single species. An analysis of restriction fragment length polymorphisms (RFLPs) with three cloned hybridization probes demonstrated that each of the P. s. tomato and P. s. syringae strains was unique. A method was developed to quantify the RFLP difference between pairs of strains, and cluster analysis revealed relationships among P. s. tomato, but not among P. s. syringae, that were similar to those based on enzyme polymorphisms. Implications of these findings for bacterial systematics and epidemiology are discussed.
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Distribution of Phthiocerol Diester, Phenolic Mycosides and Related Compounds in Mycobacteria
More LessAmong 28 mycobacterial species studied, only Mycobacterium tuberculosis, M. bovis, M. africanum, M. marinum, M. kansasii, M. gastri and M. ulcerans produced waxes yielding long-chain -diol components (called phthiocerol and companions) and polymethyl-branched fatty acids on saponification. The same mycobacterial species also produced diesters of phenol phthiocerol and companions. Fatty acids esterifying these fatty alcohols in M. marinum and M. ulcerans were found to belong to the phthioceranic series (dextrorotatory fatty acids), in contrast to those of the other species (laevorotatory fatty acids called mycocerosic acids), both groups having the same chain length and methyl-branched positions. M. kansasii and M. gastri contained the same waxes with identical structures, as did M. tuberculosis, M. bovis and M. africanum. Neither the type strain of M. tuberculosis, nor that of M. bovis or M. marinum accumulated the strain-specific phenolic glycolipids.
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