- Volume 134, Issue 6, 1988
Volume 134, Issue 6, 1988
- Genetics And Molecular Microbiology
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Molecular Cloning, Physical Mapping and Expression of the bet Genes Governing the Osmoregulatory Choline-Glycine Betaine Pathway of Escherichia coli
More LessAn analysis of the bet genes governing the osmoregulatory choline-glycine betaine pathway of Escherichia coli was performed. A 9 kb BamHI fragment, located 30 to 39 kb counterclockwise of the EcoRI site of lacZ, coded for all known Bet activities. The following genes were identified: the betA gene for the choline dehydrogenase, the betB gene for the betaine aldehyde dehydrogenase, and the betT gene or operon for the high-affinity choline transport. The betB and the betT genes were named in this paper, and the clockwise gene order was shown to be betA,B,T. Subcloning gave plasmids which expressed each of the three Bet activities separately. The cloned bet genes remained osmotically regulated, indicating the existence of several osmotically regulated promoters in the bet region. Salmonella typhimurium, which carried the bet region of E. coli in the broad-host-range vector pRK293 expressed the three Bet activities and displayed increased osmotic tolerance in the presence of choline.
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- Immunology
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Purification and Characterization of a 36 kDa Antigen of Mycobacterium leprae
More LessA 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47–9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.
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- Pathogenicity And Medical Microbiology
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Iron-regulated Outer-membrane Proteins of Escherichia coli Strains Associated with Enteric or Extraintestinal Diseases of Man and Animals
More LessThe SDS-PAGE patterns of the iron-regulated outer-membrane proteins from 70 strains of Escherichia coli isolated from various human and animal infections were analysed and the nature of the siderophores produced was examined. Iron-regulated 81 kDa and 74 kDa protein bands seen in SDS-PAGE gels were characterized further by immunoblotting using anti-81 kDa and anti-74 kDa (Cir) sera. The results showed considerable differences between the patterns of the iron-regulated outer-membrane proteins exhibited by the different strains. Nevertheless, three distinct and characteristic profiles, based on the most prominent bands expressed, could be identified, although not all strains produced patterns which matched with one of these. These results suggest the possibility of using the pattern of iron-regulated outer-membrane proteins expressed, as well as siderophores produced, as a new set of markers to characterize groups of pathogenic E. coli.
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Association of Treponema hyodysenteriae with Porcine Intestinal Mucosa
More LessThe association of Treponema hyodysenteriae with porcine caecal and colonic mucosal surfaces was studied by electron microscopy after orogastric inoculation of pigs with pure cultures. Examination of caecal and colonic mucosa from infected and control animals revealed that large numbers of the spirochaete were associated only with intestinal mucosal surfaces of infected animals. Further examination of the intestinal mucosa from infected pigs showed that T. hyodysenteriae colonized two sites preferentially: the mucus-filled crypts of Lieberkühn and the mucus gel covering the epithelium. Furthermore, no evidence of either specific or nonspecific adhesion to the epithelium proper was found, suggesting that penetration of, or trapping in the mucus gel may be the predominant mechanism of mucosal association by T. hyodysenteriae. Moreover, T. hyodysenteriae was also observed to be highly motile in intestinal mucus, moving faster than any other organism present, and this ‘high speed’ motility appeared to facilitate penetration into the mucosa. The pattern of motility observed was also highly suggestive of chemotaxis, and this was subsequently confirmed using an in vitro assay to porcine mucus material. It is suggested, therefore, that motility and chemotaxis are important factors/mechanisms in the association and colonization of porcine intestinal mucosa by T. hyodysenteriae.
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The Effect of Hormones on Trichomonas vaginalis
More LessThe hormonal milieu can alter susceptibility to infection. The effect of hormones on Trichomonas vaginalis was studied utilizing axenically cultured clinical isolates. Oestrogens, in physiological concentrations, decreased the growth of the organisms and their attachment to mammalian cells in vitro, and acted as a chemorepellent. The specificity of these effects was verified by their being blocked with anti-oestrogens, by the dose- and time-dependency of the responses, and by the lack of effect with other hormones. These results suggest that oestrogens may decrease the virulence of T. vaginalis; however, interactions between oestrogens and mammalian cells may promote the development of infection. Thus complicated interactions between hormones, micro-organisms and mammalian cells must determine whether exposure to oestrogens predisposes to or prevents the development of infection.
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Mouse Cachexia Induced by Trehalose Dimycolate from Nocardia asteroides
More LessTrehalose dimycolate (TDM) isolated from Nocardia asteroides induced in mice a severely wasted condition known as cachexia. Intraperitoneal injection of mice with five 10 μg doses of TDM in mineral oil at intervals of 2 d killed 90% of the animals within 26 d. Death followed a precipitous weight loss and an inflammatory process in the peritoneal cavity. When mice were injected intraperitoneally with a single 10 μg dose of TDM, 48 h later, they had begun to lose weight and exhibited extreme hypertriglyceridaemia and hypoglycaemia. Tumour necrosis factor (or cachectin) was detected in the plasma from animals injected with TDM. This cytokine released by mononuclear phagocytes may be involved in the induction of cachexia by TDM.
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Immunobiological Properties of Lipopolysaccharides Isolated from Fusobacterium nucleatum and F. necrophorum
More LessLipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the ‘LPS-nonresponsive’ C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.
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- Physiology And Growth
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Isolation and Physiological Characterization of Autotrophic Sulphur Bacteria Oxidizing Dimethyl Disulphide as Sole Source of Energy
More LessThe isolation of a number of strains of bacteria able to grow on dimethyl disulphide and dimethyl sulphide as sole source of energy is described. The isolates came from diverse habitats, including soil, peat, marine mud and a freshwater pond. The isolates were morphologically and physiologically best described as thiobacilli, capable of growth as Calvin cycle autotrophs on inorganic sulphur compounds, methylated sulphides or thiocyanate. They could not grow heterotrophically or methylotrophically. One isolate (E6) was examined in detail. Substrate oxidation kinetics indicated that methanethiol, sulphide, formaldehyde and formate, but not dimethyl sulphide, could be implicated as intermediates in dimethyl disulphide metabolism. Apparent K s values for the oxidation of dimethyl disulphide and methanethiol were 2.5 and 3·2 μm respectively. Growth yields in chemostat culture on dimethyl disulphide with and without thiosulphate indicated that energy conservation was probably coupled to the oxidation of formaldehyde and sulphide (derived from dimethyl disulphide via methanethiol) to CO2 and sulphate. Maximum growth yield (Y max) on dimethyl disulphide was 17 g cell-carbon per mol of dimethyl disulphide. At one dilution rate (0·078 h−1), the biomass of a culture limited by dimethyl disulphide increased when thiosulphate was also supplied, indicating a thiosulphate-dependent yield of 2·45 g cell-carbon mol−1. This is the first demonstration of the isolation of organisms into pure culture that are capable of growth on dimethyl disulphide as sole energy substrate, and of degrading it completely to CO2 and sulphate.
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Attraction of Agrobacterium tumefaciens C58C1 towards Sugars Involves a Highly Sensitive Chemotaxis System
More LessMotility of Agrobacterium tumefaciens C58C1 consisted of long straight runs, with relatively few tumbles. Speeds of up to 60 μm s−1 and runs of up to 500 μm were recorded. The propulsive mechanism appeared to resemble that of Rhizobium. Chemotaxis towards carbohydrates resolved four groups of sugars: chemoattractants with peaks at 10−6 m (sucrose, glucose and fructose); 10−5 m (maltose, lactulose and galactose); 10−4 m (raffinose, stachyose and arabinose); and weak or non-attractants (palatinose, lactose, cellobiose and xylose). In descending order, the magnitude of the responses was as follows: sucrose ⪢ maltose > lactulose > glucose > galactose/fructose > stachyose/arabinose/raffinose. The amino acids valine and arginine were good chemoattractants with peaks at 10−3 m, but no significant attraction was observed with alanine, cysteine, methionine or glycine. These results are indicative of a highly sensitive chemotaxis system towards sugars in A. tumefaciens C58C1, and suggest a role for this process in the ecology of the organism.
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C4-Dicarboxylate Metabolism in Free-living and Bacteroid Forms of Rhizobium leguminosarum MNF3841
More LessThe transport and catabolism of C4-dicarboxylic acids have been further studied in Rhizobium leguminosarum MNF3841. Uptake of [14C]succinate was induced in free-living cells of strain MNF3841 within 12 min of exposure to succinate and reached a maximum rate within 60 min. Free-living cells of strain MNF3841 oxidizing fumarate accumulated pyruvate when treated with arsenite, an inhibitor of pyruvate dehydrogenase. Generation of pyruvate from C4-dicarboxylates was accomplished by malic enzyme. Although malic enzyme was present in free-living cells grown on sucrose, higher activities were observed when fumarate or l-arabinose was the growth substrate. In crude extracts, malic enzyme activity required either NAD+ or NADP+ as cofactor, together with Mn2+, and was stimulated by K+.
Bacteroids from pea nodules transported [14C]succinate immediately after isolation, and contained twofold higher activities of both malic enzyme and pyruvate dehydrogenase than those found in fumarate-grown free-living cells. These data indicate that C4-dicarboxylates are available to nodule bacteroids and that they are catabolized through the tricarboxylic acid cycle using malic enzyme and pyruvate dehydrogenase to generate acetyl-CoA.
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Sterol Synergism in Paramecium tetraurelia
More LessParamecium tetraurelia is a naturally occurring sterol auxotroph with an absolute nutritional requirement for one of a small group of structurally related phytosterols. We report here a quantitative study demonstrating that a low, otherwise sub-supportive, concentration (≈0·020–0·050 g ml−1) of an essential phytosterol (stigmasterol) is adequate for growth of this ciliate, provided that a second, relatively non-specific sterol is available at a higher concentration (1·0 g ml−1) to allow for membrane biosynthesis. This phenomenon, referred to as sterol synergism, has been observed in a broad taxonomic range of organisms, with the conclusion that small amounts of specific sterols are required to perform some previously unknown, vital metabolic or regulatory function. Paramecium promises to be an excellent model organism for the elucidation of essential sterol function.
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A Haemoprotein Is Not Involved in the Control by Oxygen of Enteric Nitrogenase Synthesis
More LessStrains of Escherichia coli containing the Nif+ plasmid pRD1 were used to investigate the possibility that haem proteins are involved in the regulation by O2 of nif expression. Strains lacking 5-aminolaevulinate synthase (HemA−), and hence normally unable to synthesize haem proteins, showed an identical response to O2 in the presence or absence of added aminolaevulinate (and hence of haem proteins). It was concluded that the regulatory protein NifL is not a haem protein.
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Presence of Choline in Teichoic acid of Clostridium acetobutylicum N1-4 and Choline Inhibition of Autolytic Functions
More LessAddition of choline to growing Clostridium acetobutylicum led to abnormal cell septation, lack of cell separation and the consequent formation of chains. Similar results were obtained with the wild-type strain N1-4 and its autolysin-deficient mutant N1-4081. With strain N1-4, addition of choline at 1 to 2 mg ml−1 resulted in inhibition of autolysis assessed as autoplast formation in 0·6 m-sucrose, lysis by 0·3 m-NaCl/0·03 m-sodium citrate, lysis by 0·1% Triton X-100 and lysis by penicillin G. In vitro data confirmed the inhibition by choline of wall-degrading activity, using N1-4 cell walls as substrate. Choline was shown to be a component of the teichoic acid of C. acetobutylicum N1-4.
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Synthesis of Membrane and Periplasmic Proteins during Starvation of a Marine Vibrio sp
More LessChanges in membrane and periplasmic protein profiles induced by starvation conditions in the marine Vibrio sp. S14 were examined by one-dimensional gel electrophoresis. Analysis by densitometry resolved at least six periplasmic proteins, nine outer membrane proteins, and four cytoplasmic membrane proteins induced at various times during 120 h of nutrient and energy starvation. Eight of these were also synthesized by heat- and/or ethanol-shocked cells. Pulse-labelling indicated that the starvation-induced proteins were not products of degradation, and that their synthesis was differently modulated during starvation. The most pronounced changes occurred during the initial hours of nutrient and energy deprivation. The correlation between the initial changes in protein composition and utilization of the intracellular energy reserve poly-β-hydroxybutyrate is discussed. The rate of proteolysis during the initial hours of starvation was approximately 16 times greater than that during exponential growth.
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Effect of Growth Temperature on the Lipid Composition of two Strains of Thermus sp
More LessGrowth-temperature-dependent alterations in the total extractable lipid and polar lipid components of two strains of Thermus sp. isolated from a Portuguese hot spring were studied between 50°C and 78°C. The total extractable lipid varied between 8·0 and 10·6% of the cell dry weight; there were no alterations in the phosphorus and carotenoid content of the lipid extract, but the carbohydrate content increased as the growth temperature was raised. Three glycolipids and the four phospholipids were separated by thin-layer chromatography. The relative concentration of the major glycolipid (GL1) of strain SPS 17 (yellow-pigmented) increased with the growth temperature, whereas the relative concentration of a minor glycolipid (GL2) decreased. There were no temperature-dependent alterations in the relative concentrations of GL1 and GL2 from strain SPS 11 (colourless). The proportion of the major phospholipid (PL2) decreased in both strains as the growth temperature increased, whereas that of a minor phospholipid (PL1) increased. The major fatty acyl chains of both strains were 13-methyltetradecanoate (iso-C15) and 15-methylhexadecanoate (iso-C17). The GL1 in strains SPS 11 and SPS 17 had a glucose:glucosamine:glycerol:long fatty acyl chain ratio of 3:1:1:3.
† Present address: Departamento de Bioquimica, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain.
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Water Stress Plating Hypersensitivity of Yeasts: Protective Role of Trehalose in Saccharomyces cerevisiae
More LessWater stress plating hypersensitivity was studied in two strains of Saccharomyces cerevisiae, one of them being a mutant incapable of accumulating trehalose to significant levels. The wild-type strain was grown in a defined medium with glucose, maltose or ethanol as carbon/energy source. In each case plating hypersensitivity was demonstrated and resistance to the stress developed in the second half of the exponential growth phase. Development of resistance was accompanied by accumulation of trehalose and was apparently unrelated to glycerol content which, under these conditions, was always low. A qualitatively similar trend was observed in the mutant grown on glucose but trehalose levels remained low and recovery of stress resistance was only slight. Dinitrophenol induced trehalose breakdown in resting yeast and simultaneously induced the onset of plating hypersensitivity. A negative correlation was demonstrated between trehalose content and ‘plating discrepancy’ (log colony count on ‘normal’ agar – log colony count on stressing agar) for both strains under all experimental conditions. The correlation held for trehalose contents up to about 50 mg (g dry yeast)−1, above which the yeasts were apparently fully resistant. Trehalose was evidently a more effective compatible solute, per mole, than glycerol.
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The Rate and Topography of Cell Wall Synthesis during the Division Cycle of Escherichia coli Using N-Acetylglucosamine as a Peptidoglycan Label
More LessThe rates of synthesis of peptidoglycan and protein during the division cycle of Escherichia coli were measured by the membrane elution technique using cells differentially labelled with N-acetylglucosamine and leucine. During the first part of the division cycle the ratio of the rates of protein and peptidoglycan synthesis was constant. The rate of peptidoglycan synthesis, relative to the rate of protein synthesis, increased during the latter part of the division cycle. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Prior to the start of constriction the cell surface increases only by lateral wall extension. After cell constriction starts, the cell surface increases by both lateral wall and pole growth. The increase in surface area is partitioned between the lateral wall and the pole so that the volume of the cell increases exponentially. No variation in cell density occurs, because the increase in surface allows a continuous exponential increase in cell volume that accommodates the exponential increase in cell mass. The results are consistent with the constant density of the growing cell and the surface stress model for the regulation of cell surface synthesis. In addition, the elution pattern suggests that the membrane elution method does work by having the cells effectively bound to the membrane by their poles.
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- Systematics
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Outer Membrane Protein Pattern of Eubacterium plautii
More LessThe outer membrane SDS-PAGE pattern of Eubacterium plautii was characterized by a large number of surface exposed low- and high-molecular-mass proteins. Silver stainable carbohydrate was not present. The pattern was clearly distinct from those of outer membrane preparations of Eubacterium saburreum and Fusobacterium nucleatum. The results are compatible with a Gram-positive cell wall structure in E. plautii.
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Isolation and Identification of Thermotolerant Yeasts from Australian Sugar Cane Mills
More LessThermotolerant yeasts, many with the ability to ferment sucrose at temperatures above 40°C, were isolated from Queensland (Australia) cane sugar mills. Identification tests were done on 44 isolates and diagnostic keys were used to identify them. Of the thermotolerant isolates, 35 were identified as Kluyveromyces marxianus var. marxianus. Other thermotolerant strains were identified as Hansenula polymorpha, Geotrichum capitatum, Saccharomyces cerevisiae, Candida spp. and Debaromyces spp. Additional tests were done on these and on seven reference strains. Data for 75 characters for 51 strains were analysed using the simple matching coefficient and average linkage clustering. Six phenons were recovered that corresponded to the species named above.
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A Comparative Study of the Sugar Composition of Lipopolysaccharides Isolated from Vibrio cholerae, ‘Vibrio albensis’ and Vibrio metschnikovii
More LessA comparative study was made of the quantitative sugar composition of lipopolysaccharides (LPS) isolated from Vibrio cholerae (O1 and non O1 groups), ‘V. albensis’, ‘V. proteus’ and V. metschnikovii. The amino sugars 4-amino-4,6-dideoxy-d-mannose (perosamine) and 2-amino-2,6-dideoxy-d-glucose (quinovosamine) were present exclusively in LPS isolated from S-form O1 group of V. cholerae regardless of serotype (i.e. Ogawa or Inaba) and biotype (i.e. classical or eltor). Classical O1 group V. cholerae was distinguishable from eltor O1 group V. cholerae on the basis of the fructose content of the LPS: >3% and ≤1%, respectively. Distinct differences in the sugar composition of LPS were observed between V. cholerae and ‘V. albensi’, ‘V. proteus’ and V. metschnikovii.
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