- Volume 137, Issue 7, 1991
Volume 137, Issue 7, 1991
- Pathogenicity And Medical Microbiology
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Purification of Chlamydia trachomatis by a simple and rapid filtration method
More LessSummary: A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0·6 μm pore diameter polycarbonate filter. The filtrate was then passed through a 0·2 μm pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.
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Growth of Haemophilus influenzae type b in the presence of bovine aortal endothelial cells
More LessSummary: In serum-free medium in the presence of bovine aortal endothelial cells (BAOEC), Haemophilus influenzae type b was capable of extensive proliferation compared to that in serum-free medium alone. An unidentified lowmolecular-mass (< 2000 kDa) compound(s) was, in part, responsible for this phenomenon. There were changes in the outer-membrane protein profiles between broth-grown (the original inoculum) and BAOEC-grown organisms, particularly in the 45-70 kDa range. Both broth-and BAOEC-grown bacteria were serum sensitive in vitro but could be converted to a serum-resistant phenotype, resembling that found in vivo, by incubation in a serum filtrate.
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The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34
More LessSummary: The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype O:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serum-resistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype O:34 with moderate virulence.
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Adhesion of K99 fimbriated Escherichia coli to pig intestinal epithelium: correlation of adhesive and non-adhesive phenotypes with the sialoglycolipid content
More LessSummary: Evidence for the existence of two phenotypes of piglets born to experimental herds was obtained based on the susceptibility of intestinal brush borders to adhesion of K99-positive Eschenchia coli. The enterocytes of the K99-receptive piglets displayed a characteristic sialoglycolipid pattern, with a higher content of the monosialoglyco-lipids II3 NeuGc-LacCer (GM3Gc), IV3NeuGc-nLcOse4Cer (SPGGc) and IV3NeuAc-nLcOse4Cer (SPG) and the oligosialogangliosides IV3NeuAc, II3NeuAc-GgOse4Cer (GD1a), II3(NeuAc)2-GgOse3Cer (GD2), II3 (NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc, II3(NeuAc)2-GgOse4Cer (GT1b) when compared to the gangliosides of non-receptive piglets. The gangliosides from enterocytes of the non-receptive piglets were mainly the monosialogangliosides II3NeuAc-GgOse3Cer (GM2) and II3NeuAc-LacCer (GM3), only traces of the other sialoglycolipids being detected. Adhesion of 14C-labelled K99-positive E. coli cells to the piglet small intestinal sialoglycolipids, as tested by the thin-layer chromatogram overlay assay, revealed that the receptive enterocyte membrane was richer in glycolipids containing K99 receptor structures than the non-receptive enterocyte. Adhesion of K99-positive E. coli correlated with the degree of sialylation of the brush border glycolipids.
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Different promoters of SHV-2 and SHV-2a β-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers
More LessSummary: Clinical Klebsiella pneumoniae isolates as well as Escherichia coli transformants producing the ß-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l−1 or 64 to >128 mg l−1, respectively. The ß-lactamases differ by one possibly insignificant amino acid exchange at position number 10 of the mature protein; their kinetic parameters are rather similar. The 5' untranslated regions of both corresponding genes show no homology starting 74 nucleotides upstream to the start codon. Hybridization of intragenically annealing oligonucleotides to dot-blotted serial dilutions of total cellular RNA from E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1·2 μg (SHV-2) and 0·32 to 0·16 μg (SHV-2a), indicating a four to eight times higher amount of specific transcript in the case of SHV-2a. By primer extension analysis and S1 nuclease digestion the starting point of transcription was located 100 nucleotides (SHV-2) and 50 nucleotides (SHV-2a) in front of the start codon. No other transcripts of different length could be detected after prolonged exposure. Northern blot analysis demonstrated the length of the ß-lactamase mRNA to be about 1·6 kb in both cases, thus comprising a potential open reading frame downstream of the two enzymes’ genes. Selective PCR amplification of both promoter regions and of the structural gene of SHV-2 and subsequent combined cloning of each of the promoters and the SHV-2 gene into pBGS19 using a BamHI restriction site introduced by three point mutations into the cloned sequences was employed to transform E. coli DH5a. The MIC value for cefotaxime of transformants harbouring the SHV-2 promoter and SHV-2 structural gene was 8 mg l−1, but was 64 mg l−1 in the case of the SHV-2a promoter and SHV-2 structural gene combination, indicating that the quantitative change of resistance to cefotaxime of SHV-2a-producing bacteria is caused solely by a significant promoter mutation.
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- Physiology And Growth
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Growth kinetics of Agaricus bisporus mycelium on solid substrate (mushroom compost)
Growth of Agaricus bisporus was studied on sterile compost and on sterile compost pre-grown with the thermophilic fungus Scytalidium thermophilum. Early growth was characterized by depressed, slowly growing hyphae. The mycelial radius extended exponentially in this stage. Mycelium on sterile compost switched to a fluffy growth type of radially orientated hyphae that extended at a linear rate, K r, of 5 mm d−1. On compost pre-grown with S. thermophilum, early growth continued up to 30 mm, then extension became linear at K r = 7·2 mm d−1. If cultures were well ventilated, K r was higher. A new growth function was derived to combine both growth phases. The width of the peripheral growth zone, w, of A. bisporus mycelium was 3 mm. The exponential specific growth rate, μ, was calculated from K r/w = 2·4 d−1, which is much higher than a previous estimate of 0·19 d−1. In addition to growth-promoting effects, S. thermophilum had inhibitory effects; the period of early growth was reduced if S. thermophilum was inactivated in pre-grown compost. Under commercial conditions, maximum growth rates are not exploited. The introduction of selected strains of thermophilic fungi in compost and the development of a new kind of spawn inoculum of A. bisporus might lead to improvements in commercial practice.
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Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture
More LessKlebsilla pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-Iimited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.
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The increase of KCN-sensitive electron flow in the branched respiratory chain of Micrococcus luteus grown slowly in carbon-limited culture
More LessSummary: The strictly aerobic bacterium Micrococcus luteus was grown in the presence of lactate as sole carbon source under conditions of excess substrates (in batch culture) or under strict lactate (C) or ammonium (N) limitation (in a chemostat, D = 0·02 h−1). KCN (2mm) stimulated the respiration of batch-grown and N-limited cells, and inhibited the oxidase activity of C-limited cells by 40–60%. The content and composition of cytochromes and the KCN-dependences of the oxidase activities were found to be the same for the membranes of C-limited and batchgrown cells. The KCN sensitivity of the oxidase activities of isolated membranes decreased in the order NADH > malate > lactate. NAD-dependent lactate and malate dehydrogenase activities were observed in the cytoplasm of C-limited cells but not in that of batch-grown cells. The stimulation of cell respiration by lactate, maiate, pyruvate or ethanol caused a marked increase of the steady-state level of NAD+ reduction only in C-limited cells. It is assumed that the slow growth of C-limited cells is accompanied by an increase in the formation of NADH, which is oxidized by the KCN-sensitive, tightly-coupled, main branch of the M. luteus respiratory chain. Under non-limited conditions of growth the respiration is due mainly to the direct oxidation of lactate via the weakly coupled alternative branch. The role of this switching of the electron flow from one pathway to the other during the adaptation of the bateria to the slow C- and energy-limited growth is discussed.
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Fatty acid composition of the estuarine Flexibacter sp. strain Inp: effect of salinity, temperature and carbon source for growth
More LessSummary: The total fatty acid content of the estuarine Flexibacter sp. strain Inp and the relative proportions of its constituent fatty acids were affected by growth temperature and salinity. Whilst both the proportion and concentration of the polyunsaturates were markedly stimulated by increases in salinity, the total amount of fatty acid per mg cell protein decreased. The highest concentration of fatty acid per mg cell protein did not coincide with the highest percentage of polyunsaturated fatty acids, which occurred when the bacterium was grown on glucose. The presence of an inverse relationship between C16: 1w5 and C18: 1w9 are regarded as evidence that two different pathways exist for the biosynthesis of unsaturated fatty acids in Flexibacter strain Inp.
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Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus
More LessSummary: Cephalosporin biosynthetic activity and extracellular protease production increased during growth of Streptomyces clavuligerus in defined medium, while the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) remained very low and stable. Cephalosporin biosynthesis (measured in resting cell systems) was initiated during early exponential growth in complex media, without appreciable change in the small ppGpp pool. Nutritional shift-down induced by withdrawal of Casamino acids caused a transient increase in ppGpp and a reduction of RNA accumulation. The increase in ppGpp was small in very young cultures, but increased as the culture aged. Twenty-seven spontaneous thiostrepton-resistant mutants were isolated and partially characterized. Most of them had a reduced ppGpp-forming ability and gave normal titres of cephalosporin. However, in complex medium, some mutants did not produce cephalosporins or extracellular protease, whereas others overproduced cephalosporins. The results indicate that, in S. clavuligerus, there is no obligatory relationship between the initiation of secondary metabolism and the stringent response.
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l-Arginine is essential for the formation in vitro of ethylene by an extract of Pseudomonas syringae
More LessSummary: A system was developed for the formation of ethylene in vitro by an extract of Pseudomonas syringae pv. phaseolicola PK2. The ethylene-forming activity of a cell-free extract of this bacterium measured in a system reported previously was almost completely lost when the cell-free extract was dialysed against potassium phosphate buffer for 24 h at 4 °C. When the fraction of cell-free extract with a molecular mass < 10 kDa (SupI) was added back to the enzyme fraction after gel filtration of the cell-free extract, the enzymic activity increased to about four times that of the gel-filtered crude enzyme. The action of SupI could be reproduced by the addition of l-arginine. The complete system for the formation of ethylene under aerobic conditions in vitro required 0·25 mm-2-oxoglutarate, 0·2 mm-FeSO4, 2 mm-DTT, 10 mm-l-histidine and 0·2 mm-l-arginine. The cofactor specificity was examined by replacing l-arginine or l-histidine with various analogues, but none of them were effective. The components of this system, with the exception of l-histidine, were similar to those of a system derived from the ethylene-producing, plant pathogenic fungus Penicillium digitatum which also produced ethylene in vitro in a reaction dependent on 2-oxoglutarate. The intermediates in the ethylene-forming reaction are postulated and the roles of l-arginine and l-histidine in the formation of ethylene by Ps. syringae are discussed.
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Production of protoplasts of Penicillium cyclopium with improved viability and functional properties
More LessSummary: A new simple procedure for the production of protoplasts of Penicillium cyclopium with high regeneration rates and efficient transport activity is described, involving the use of a preparation of Novozym 234 with very low protease activity. The combination of a heat pretreatment at 55 °C for 15 min with the use of the protease inhibitor aprotinin resulted in a 97% reduction of Novozym 234 protease activity with respect to untreated controls. Polysaccharide-hydrolysing activity was inhibited much less, to 60% of the untreated Novozym 234 level. Protoplasts could be successfully produced with the new low-protease Novozym 234 preparation, showing a threefold increase in regeneration capacity compared to control protoplasts obtained with the original preparation. The rates of 3-O-methylglucose uptake and the capacity to accumulate this sugar analogue were also higher in protoplasts obtained by the new method.
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Evidence for a compartmentation of penicillin biosynthesis in a high- and a low-producing strain of Penicillium chrysogenum
More LessSummary: Pulse-chase experiments using [U14-C]valine were done with P2 and Q176, high- and low-penicillin-producing strains of Penicillium chrysogenum. The metabolic flux of this amino acid into protein and penicillin was measured, and compartmentation of penicillin biosynthesis was assessed. Strain P2 took up 14C-valine more slowly than strain Q176, but their rates of incorporation into protein were comparable. Incorporation of 14C-valine into penicillin occurred immediately with the high-producer P2, but exhibited a lag with Q176. After 14C-valine had been removed from the medium, the specific radioactivity of penicillin continued to increase in Q176 but started to decrease immediately in P2. The specific radioactivities of 14C-valine in protein and in penicillin were significantly different in both strains: Q176 had a higher specific radioactivity of valine in penicillin than P2, whereas P2 had a higher specific radioactivity of valine in protein than Q176. Moreover, the specific radioactivity of 14C-valine in penicillin was 20-fold higher in strain Q176 than in P2. These results indicate that penicillin and protein biosynthesis use different pools of cellular valine, and that exchange of valine between the two compartments is slow in the low-producer, but rapid in the high-producer strain. Hence these results indicate a further control point of penicillin biosynthesis in P. chrysogenum.
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Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of ß-lactam antibiotic
More LessSummary: Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of ß-bactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the ‘random addition’ model for multiple cross-link formation and not all those predicted by the ‘monomer addition’ model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.
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Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH
More LessSummary: Incubation of Saccharomyces cerevisiae at sub-lethal temperatures results in an increase in thermotolerance. This process is dependent not only on the sub-lethal temperature but also on the duration of sub-lethal heating. This indicates that the mechanism inducing thermotolerance is a time/temperature dose response. Other factors that induce thermotolerance include exposure to ethanol, sorbic acid and low external pH values. These factors induce thermotolerance after incubation in the presence of protein synthesis inhibitors, and they are all known to affect the intracellular pH (pHi). The acquisition of increased thermotolerance is minimal with sub-lethal heating under neutral external pH conditions. However, when the external pH is reduced to 4·0 the level of induced thermotolerance increases to a maximum value. Using a specific ATPase inhibitor, diethylstilboestrol (DES), ATPase activity was shown to be essential for the cell to survive heat stress. In addition, measurement of acid efflux, or ATPase activity, revealed that proton pumping from the cell increased by approximately 50% at sublethal temperatures that induce thermotolerance. This work has clearly implicated pHi perturbation as the triggering mechanism conferring thermotolerance on S. cerevisiae.
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Role of Na+ in pH homeostasis by the alkalophilic bacterium Exiguobacterium aurantiacum
More LessSummary: Exiguobacterium aurantiacum, a facultative alkalophile, can maintain a dpH of up to 1·7 pH units, acid inside, and can rapidly adjust the cytoplasmic pH (pHi) in response to a shift in external pH (pHo), demonstrating effective pHi homeostasis. The presence of Na+ accelerated the attainment of a new steady-state pHi during a shift in the alkaline direction but slowed the attainment of new steady state following a shift in pHo in the acid direction. Measurements of internal Na+ following the addition of 6 mm-NaCl to cells incubated under conditions whereby the cells either could (+ 0·68 mm-NaCl) or could not (0·08 mm-NaCl) regulate pHi indicated that pHi exerted some feedback control over Na+ influx. A model for the involvement of Na+ in pHi regulation comprising an electrogenic Na+/H+ antiporter and a sodium influx channel regulated by pHi is proposed. Intrinsic to this model is the suggestion that the Na+/H+ antiporter is not the sole site of feedback control by pHi.
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The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat
More LessSummary: Streptomyces thermoviolaceus was grown in a chemostat under conditions of glutamate limitation. The effects of growth rate on production of the antibiotic granaticin, extracellular protein and protease activity as components of secondary metabolism were studied at 37, 45 and 50 °C. The amount of each secondary metabolite synthesized was highly dependent on growth rate and temperature. Granaticin yields were highest at growth rates of 0·1 to 0·15 h−1 at 37 °C, 0·175 h−1 at 45 °C and 0·045 h−1 at 50 °C. Protease activity of culture supernatants responded to low nutrient concentration and/or low growth rate. Measurements of extracellular protein revealed complex changes in amount which were dependent on growth rate and temperature. At 45 °C and a growth rate of 0·15 h−1, biomass yield was highest between pH 5·5 to 6·5 whereas granaticin synthesis was low at pH 5·5 and rose to highest values at between pH 6·5 and 7·5.
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The parasitic flagellates Trichomonas vaginalis and Tritrichomonas foetu produce indole and dimethyl disulphide: direct characterization by membrane inlet tandem mass spectrometry
More LessSummary: The use of a membrane inlet triple quadrupole mass spectrometer revealed indole as an end product in the growth medium of cultures of the cattle parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis: formation of indole is enhanced in the presence of added tryptophan. Two different clinical isolates of Trich. vaginalis also produce dimethyl disulphide. Electron impact ionization yielded complex fragmentation mixtures, but the facility for analysis of daughter ions enabled unequivocal assignments. Chemical ionization gave [M + 1]+ species, and tandem mass spectrometry produced identification through daughter ions. The method provides a rapid single-step procedure for the characterization of microbial products without the need for preliminary separation and derivatization.
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Analysis of the response of Saccharomyces cerevisiae cells to Kluyveromyces lactis toxin
More LessThe response of Saccharomyces cerevisiae cells to the toxin produced by certain strains of Kluyveromyces lactis was studied. The toxin caused an arrest of sensitive cells in the unbudded (G1) phase of the cell cycle, consistent with the accumulation of cells with an unreplicated (G1) content of DNA in treated populations. However, toxin-treated cells were not proficient for mating. The effects of the toxin were dependent on its continuous presence for over an hour and removal of cells into fresh medium at earlier times prevented inhibition. Following toxin treatment, cells increased in volume and continued to synthesize protein and RNA, suggesting that they were able to continue growth in the absence of division. However, several lines of evidence suggested that the toxin does not simply block proliferation in G1, but that another continuous or post-G1 event is also affected. Possible models to explain these observations are discussed.
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- Systematics
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Grouping of Xanthomonas campestris pathovars by SDS-PAGE of proteins
More LessSummary: A numerical analysis was performed on SDS-PAGE protein patterns of 307 Xanthomonas strains comprising all species and 27 X. campestris pathovars. The electrophoretic groupings corresponded in some, but not all cases with the existing pathovars. Six pathovars constituted distinct entities, comprising all strains investigated. These included X. campestris pv. campestris, X. campestris pv. graminis, X. campestris pv. hyacinthi, X. campestris pv. pelargonii, X. campestris pv. pruni and X. campestris pv. theicola. A great number of pathovars consisted of a homogeneous group from which only one or a few strains were aberrant. In two cases (X. campestris pv. ricini and X. campestris pv. vitians) even the pathovar reference strain was aberrant. Six pathovars from members of the plant family Fabaceae could not be differentiated from one another: X. campestris pv. phaseoli, X. campestris pv. phaseoli var. fuscans, X. campestris pv. cajani, X. campestris pv. vignicola, X. campestris pv. alfalfae and X. campestris pv. glycines. At least six X. campestris pathovars were heterogeneous, displaying two or more protein electrophoretic types: X. campestris pv. alfalfae, X. campestris pv. dieffenbachiae, X. campestris pv. juglandis, X. campestris pv. poinsettiicola, X. campestris pv. vesicatoria and X. campestris pv. vignicola. A database of SDS-protein patterns provides a valuable tool for the identification of unknown xanthomonads.
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