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Volume 140,
Issue 4,
1994
Volume 140, Issue 4, 1994
- Pathogenicity And Medical Microbiology
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Chemical and immunological analysis of the Aspergillus fumigatus cell wall
More LessHyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1·3)-α-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6·2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-β-d-galactofuranosidase. The alkaliinsoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1·3)-β-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin.
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Variation in outer-membrane protein and lipopolysaccharide profiles of Pasteurella haemolytica isolates of serotypes A1 and A2 obtained from pneumonic and healthy cattle
More LessThe outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 29 isolates of Pasteurella haemolytica serotypes A1 (18 isolates) and A2 (11 isolates), obtained from pneumonic (13 isolates) or healthy (16 isolates) cattle, were compared by SDS-PAGE and Western blot analysis. Coomassie-blue-stained OMP profiles of serotype A1 and A2 isolates could be distinguished from each other by differences in both major and minor proteins. Whereas the OMP profiles of the serotype A1 isolates were extremely uniform in stained gels, there was variation in the mobilities of high-molecular-mass minor proteins and one of the major proteins of serotype A2 isolates. Differences in the OMP profiles of isolates within both the A1 and A2 serotypes were more clearly distinguished by Western blotting than by staining after SDS-PAGE. Thus, by Western blot analysis, four distinct OMP profiles were identified within the serotype A1 and A2 isolates, respectively. The profiles of the serotype A1 isolates were designated OMP types 1.1, 1.2, 1.3 and 1.4; those of the serotype A2 isolates were designated OMP types 2.1, 2.2, 2.3 and 2.4. Three distinct LPS profiles were recognized among the isolates which, by comparison with previously described LPS types, were identified as smooth LPS type 1 and rough LPS types 3 and 5. Isolates of serotype A1 consisted of LPS type 1 only, whereas isolates of serotype A2 consisted of LPS types 3 or 5. OMP and LPS analysis of P. haemolytica has applications in epidemiological and virulence studies.
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Phospholipids of Mycobacterium intracellular inhibit T cell blastogenesis
More LessA crude lipid fraction obtained from Mycobacterium intracellulars (MI whole lipids) suppressed concanavalin A (Con A)-induced blastogenesis of murine spleen cells (SPCs). Among three lipid fractions, the phospholipid fraction possessed the highest inhibitory activity, followed by the polar mycoside fraction, but the apolar mycoside fraction showed no activity. Since MI whole lipid and phospholipid fractions inhibited the Con A-induced proliferative response of SPCs which had been treated with Con A before the addition of the lipid fractions, their action is not due to hindrance of Con A binding to T cells. These two MI lipid fractions reduced IL-2-producing ability, acquisition of IL-2 reactivity, and expression of IL-2 receptors in Con A-stimulated T cells. However, they did not affect IL-2-induced proliferation of an IL-2-dependent cytotoxic T cell line, CTLL-2. When SPCs were pretreated with either MI whole lipid or phospholipid fraction for 24 h, an irreversible reduction in Con A responsiveness was seen only in the phospholipid-treated SPCs. The action of these MI lipid fractions was phase-dependent and they exhibited considerably decreased but still significant inhibitory activity against Con A blastogenesis of SPCs, even when they were added at 24 h after the initiation of SPC culture with Con A. MI whole lipids and the three lipid fractions (polar mycoside, apolar mycoside, and phospholipid fractions) did not exhibit suppressor cellinducing activity, while MI whole lipid fraction antagonized the Con A-mediated generation of suppressor cells. Silica gel thin layer chromatography of the phospholipid fraction showed four spots containing phosphate and one spot without. SPC Con A blastogenesis-inhibitory activity was shared by the two least polar phosphate-containing substances. One of those apparently contained amino groups and carbohydrate moieties. The second component contained no such moieties.
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The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50
More LessPorphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (μmax) was determined at both haemin concentrations (μmax = 0.236 ± 0.052 and 0.271 ± 0.039 h-1 respectively). This enabled dilution rates to be adjusted so that the virulence and enzyme activity of haemin-limited and haemin-replete cells could be compared at identical relative growth rates (μrel) of 0.25, 0.50 and 0.75 of their respective μmax. The data showed that the fastest growing cells were significantly more virulent than those grown more slowly, irrespective of haemin concentration. However, at each growth rate tested, cells grown under haemin-excess conditions were always more virulent than haemin-limited cells. Trypsin-like enzyme activity of whole cultures was also greater at each growth rate under haemin-excess conditions while, conversely, collagenolytic activity was generally higher in haemin-limited cultures. Thus, although growth rate had an effect on the virulence and enzyme activity of P. gingivalis, the availability of haemin for growth was the most significant factor.
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Molecules of Streptococcus gordonii that bind to Porphyromonas gingivalis
More LessInterbacterial binding is considered an important colonization mechanism for many of the organisms that inhabit dental plaque. Porphyromonas gingivalis, a periodontal pathogen, can adhere to species that comprise early plaque, such as Streptococcus gordonii. In this study, the molecules of S. gordonii G9B that mediate binding to P. gingivalis were investigated. Biotinylated surface molecules of S. gordonii were extracted and mixed with P. gingivalis cells. Interactive streptococcal components were identified by SDS-PAGE of the P. gingivalis cells followed by electroblotting, and visualization of the adsorbed streptococcal molecules with streptavidin-alkaline phosphatase. S. gordonii molecules of 45 kDa and a doublet of 62/60 kDa were observed to bind to P. gingivalis. Polyclonal antibodies raised to the 62/60 kDa proteins inhibited the binding interaction. These antibodies demonstrated an antigenic relationship between the 62/60 kDa molecules and the 45 kDa protein. Both molecules were also antigenically related to, and may be breakdown products of, a larger molecule of 170 kDa which is antigenically related to the P1 antigen of S. mutans. Cloning and expression in Enterococcus faecalis of the gene for the P1-like molecule from S. gordonii M5 resulted in a phenotype that expressed the 62/60 kDa and 45 kDa antigens and was capable of binding to P. gingivalis. These results suggest that a P1-like molecule in S. gordonii is involved in adherence to P. gingivalis. Processing of the P1-like molecule into smaller fragments of 62/60 kDa and 45 kDa may be required for binding activity.
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Hydrolytic enzymes and lectin-binding activity of black-pigmented anaerobic rods
More LessRecent taxonomic studies on black-pigmented anaerobic rods, a group of bacteria found on mucosal surfaces of humans and animals, led to the subdivision of existing species and to the creation of new species. The aim of this study was to characterize all 11 currently recognized species of black-pigmented bacteria (55 strains) for their ability to hydrolyse a variety of natural and synthetic substrates and for their lectin reactivity. Although most of the strains demonstrated some activity against proteinaceous substrates, Porphyromonas gingivalis was the only species able to hydrolyse type I collagen. Most strains possessed glycylprolyl protease activity, elastase-like activity and phospholipase C activity, whereas trypsin-like activity was restricted to P. gingivalis, Porphyromonas salivosa and Bacteroides macacae. β-Lactamase activity was demonstrated in five strains belonging to the saccharolytic group. The lectin reactivity of the bacteria was determined by a dot-blot procedure using horseradish-peroxidase-conjugated lectins. Three lectins, LOTUS A, RCA-1 and ConA, failed to react with any of the bacteria tested. WGA reacted strongly with the cell surface of human biotypes of asaccharolytic black-pigmented bacteria (P. gingivalis, Porphyromonas asaccharolytica and Porphyromonas endodontalis) and Prevotella intermedia. The animal biotype strains of P. gingivalis showed a higher affinity for SBA and PNA than for WGA.
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- Physiology And Growth
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Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect
More LessGrowth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0·1 h−1 With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol l−1 h−1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0·1 h−1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.
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Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis
Aerobic, glucose-limited chemostat of Saccharomyces cerevisiae CBS 8066 cometabolized propionate when this compound was added to the reservoir medium. Co-metabolism of propionate led to an increase of the biomass and protein yields. Attempts to grow S. cerevisiae on propionate as a sole source of carbon and energy were not successful. Activities of propionyl-CoA synthetase in cell-free extracts were sufficient to account for the rates of propionate consumption observed in the chemostat cultures. Activities of propionyl-CoA carboxylase, a key enzyme of the methylmalonyl-CoA pathway of propionate metabolism, were negligible. In contrast, activities of 2-methylcitrate synthase, a key enzyme activity of the 2-methylcitrate pathway of propionate metabolism, increased substantially with increasing propionateto-glucose ratios in the reservoir media, and were sufficient to account for the propionate consumption rates observed in the chemostat cultures. This suggested that the 2-methylcitrate pathway is the major pathway of propionate metabolism in S. cerevisiae. In the literature, labelling patterns observed after incubation of this yeast with [3-13C]propionate have been interpreted as evidence for channelling of tricarboxylic acid (TCA) cycle intermediates, possibly as a consequence of the organization of TCA cycle enzymes in a metabolon. However, this interpretation of 13C-labelling patterns rested on the assumption that propionate metabolism in S. cerevisiae occurs via the methylmalonyl-CoA pathway. Since the distribution of 13C in alanine reported in the literature is fully compatible with a major role of the 2-methylcitrate pathway in propionate metabolism, it cannot be interpreted as evidence for the existence of a TCA cycle metabolon in S. cerevisiae.
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Analysis of the induction of general stress proteins of Bacillus subtilis
In Bacillus subtilis stress proteins are induced in response to different environmental conditions such as heat shock, salt stress, glucose and oxygen limitation or oxidative stress. These stress proteins have been previously grouped into general stress proteins (Gsps) and heat-specific stress proteins (Hsps). In this investigation the N-terminal sequences of 13 stress proteins of B. subtilis were determined. The quantification of the mRNA and the analysis of the protein synthesis pattern support the initial hypothesis that the chaperones DnaK and GroEL are Hsps in B. subtilis. In contrast, the recently described proteins GsiB, Ctc and RsbW belong to a class of Gsps that are induced by various stresses including heat shock. The main part of the Gsps described in this study failed to be induced in the sigB deletion mutant ML6 in response to heat shock. However, all the five Hsps were induced in this mutant in response to heat shock. These data indicate that SigB plays a crucial role in the induction of general stress genes, but is dispensable for the induction of Hsps.
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Regulation of xylanolytic enzymes in Bacillus subtilis
More LessThe synthesis of the xylanolytic enzymes β-xylanase and β-xylosidase of Bacillus subtilis was studied. In contrast to many catabolic extracellular enzymes, β-xylanase was synthesized constitutively during exponential growth and was not repressed by glucose. β-Xylosidase synthesis was induced 100-fold by xylose and repressed 100-fold by glucose. Carbon catabolite repression was abolished in a ccpA mutant. Titration experiments using a multicopy operator sequence respondible for carbon catabolite repression indicated that the gene encoding β-xylosidase is part of the same carbon catabolite repression regulon as the amyE and bgIS genes.
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Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics
More LessSubmerged cultures of four Streptomyces species accumulated triacylglycerol (TAG), ranging from 50 to 150 mg (I medium)−1, during the post-exponential phase of growth. S. lividans also produced glycogen (80 mg I−1). Identity of TAG species was confirmed after TLC, by mass spectroscopy and by quantitative IR spectroscopy and reaction of the hydroxamate derivatives with ferric chloride. This is the first substantive report showing the storage of TAG in bacteria. Distribution of diacylglycerol acyltransferase (TAG synthase) activity from S. coelicolor and S. lividans during incubation on different media paralleled the formation of TAG. Accumulation of TAG may be necessary to maintain cell integrity after glucose becomes exhausted from the medium, and also to provide the C2 units needed for subsequent biosynthesis of acetate-derived antibiotics in appropriate species. Actinorhodin was formed by S. coelicolor when grown on YEME medium only after exhaustion of glucose; the carbon source may therefore originate from TAG. All the organisms examined in this study formed isoprenoid-derived hydrocarbons (up to 3 mg I−1) which were identified as squalene plus hydrogenated derivatives.
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Nitrogen fixation by Plectonema boryanum has a photosystem II independent component
More LessThe non-heterocystous filamentous cyanobacterium Plectonema boryanum showed a several-fold decline in photosystem II dependent O2 evolution during the diazotrophic phase of growth. A sharp fall in the amounts of light-harvesting pigments and an uncoupling between the electron transfer from the photosystem II complex to the quinone acceptors may lead to the depression of light-dependent O2 evolution during the diazotrophic phase. Nitrogen fixation as well as CO2 fixation required light, but were partly supported independently of photosystem II. A stimulation of photosystem I and a depression of photosystem II occurred during the diazotrophic phase of a nitrogen-fixing culture of P. boryanum.
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- Plant-Microbe Interactions
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Characterization of cell surface components of Azospirillum brasilense Sp7 as antigenic determinants for strain-specific monoclonal antibodies
Monoclonal antibodies (mAbs) with high specificity for Azospirillum brasilense Sp7, and which bind to different antigenic determinants, were characterized using Western blot techniques applied to one- and two-dimensional fingerprints of the outer-membrane components, and by immunogold labelling combined with transmission electron microscopy. One class of mAbs, which bound to A. brasilense Sp7 and the closely related strain Cd, recognized a 100 kDa protein subunit of the polar flagellum. Two classes of strain-specific mAbs for A. brasilense Sp7 bound, respectively, to a 85 kDa outer-membrane protein and to polysaccharide.
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- Systematics
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Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles
More LessThe genetic diversity in Trichoderma harzianum isolates from mushroom compost was assessed using various molecular techniques. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) divided the 81 isolates into three major groups, 1, 2 and 3. There was no variation within a group in rDNA, while a low degree of polymorphism was detected in mtDNA. Random amplified polymorphic DNA (RAPD) analysis of 30 randomly chosen isolates, with six primers, in general confirmed the RFLP groups. Nucleotide sequence determination of rDNA internal transcribed spacer (ITS) 1 revealed three distinct ITS types, 1, 2 and 3, possessed by isolates from the respective groups 1, 2 and 3. Based on these molecular data, group 2 isolates, which are aggressive colonizers of mushroom compost, could be clearly distinguished from the isolates belonging to the other two groups.
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Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains
In the recently described species Campylobacter helveticus, two sizes of PCR amplicon were detected with primers homologous to conserved regions of the 16S rRNA gene. A conventionally sized gene was sequenced from the type strain, NCTC 12470, placing the new species as phylogenetically related to C. upsaliensis and the thermotolerant campylobacters. This nucleotide sequence enabled PCR primers to be designed for use in rapid molecular identification of C. helveticus and its closest phylogenetic relative, C. upsaliensis. When this assay was employed to characterize 22 ‘C. upsaliensis-like’ isolates, twelve were identified as C. helveticus and nine as C. upsaliensis, in agreement with data obtained with a C. helveticus-specific DNA probe. A 550 bp amplicon internal to the 16S rRNA gene of C. helveticus was used to determine restriction fragment length polymorphisms (RFLPs) in genomic Southern blots, confirming that the copy number of the C. helveticus gene was three, and identifying nine 16S rRNA gene profiles. In 5/12 C. helveticus isolates identified by PCR, an enlarged amplicon was detected. The enlarged 16S rRNA gene of one of these strains, NCTC 12838, was sequenced and shown to contain an atypical intervening sequence (IVS) of 148 nucleotides. The position and size of such an IVS was inferred in the other four isolates by PCR with primers 5′ and 3′ to its position in NCTC 12838. This is a first report of an IVS in the 16S rRNA gene of a eubacterium.
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Rhodobacter marinus sp. nov.: a new marine hydrogen producing photosynthetic bacterium which is sensitive to oxygen and sulphide
More LessA new non sulphur photosynthetic bacterium, strain NKPB 0021, was isolated from sea water samples and assigned to the genus Rhodobacter on the basis of morphology and presence of vesicular photosynthetic membranes. Cells are motile rods, 0.6–1.2 μm in diameter and multiply by binary fission. This isolate is unusual since it is oxygen sensitive and cannot grow aerobically. Strain NKPB 0021 grows best anaerobically in the light and requires NaCl for growth (optimum 1.5%). Cells are capable of assimilatory sulphate reduction, and are sensitive to sulphide, with no growth above 0.7 mM sulphide. The mean DNA base composition is 66.7 mol% G + C. On the basis of this study, we propose strain NKPB 0021 to be the type strain of a new species, Rhodobacter marinus.
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- Genome Analysis
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Cloning the ribosomal RNA operons of Mycoplasma flocculare and comparison with those of Mycoplasma hyopneumoniae
More LessIn contrast to other mycoplasma species the 16S/23S rRNA and 5S rRNA operons of Mycoplasma flocculare and Mycoplasma hyopneumoniae map at least 150 kb apart (20% of the genome). Both operons from M. flocculare have been cloned and sequenced. The 23S rRNA gene sequence showed 96.7% homology with the corresponding gene of M. hyopneumoniae, equalling that found earlier for 16S rRNA and confirming the close phylogenetic relationships of these organisms. A possible upstream promoter was identified. Sequence elements upstream and downstream from each structural gene could form a stem needed for maturation of the immature rRNA transcript to mature 16S and 23S rRNA. We also identified two possible stem-and-loop sequences 3' to the 23S rRNA gene. The 5S rRNA gene itself also showed high homology with the corresponding structural gene of M. hyopneumoniae, although the upstream and downstream sequences were highly heterologous.
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- Corrigendum
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