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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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