- Volume 132, Issue 3, 1986
Volume 132, Issue 3, 1986
- Pathogenicity And Medical Microbiology
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Immunoelectrophoretic Analysis of Streptococcus agalactiae Serotype Ia Antigens
More LessSUMMARY: Rabbit antibodies to heat-killed whole cells of Streptococcus agalactiae serotype Ia were used to establish an antigen map using Triton X-100 sonicates of homologous cells and crossed immunoelectrophoresis. A total of 11 antigens were identified but the density of immunoprecipitates was varied and only seven could be reliably detected, one of which dominated the immunoprecipitate pattern by its intensity. The antigens were partially characterized by immunological, chemical and cell-location methods. Five of the antigens contained carbohydrate and two of those were sensitive to trypsin and probably represent cell-wall compounds. Of the three most prominent antigens, one was surface located and represented the type and shared type antigens (Iabc), one was a cell-wall carbohydrate and very sensitive to periodate, and one was a protein/carbohydrate complex which was heat-labile and trypsin sensitive. Group B epitopes were detected in three immunoprecipitates. Cross-reactions between type Ia and other serotypes and streptococci were recorded.
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Multilocus Genotypes Determined by Enzyme Electrophoresis of Neisseria meningitidis Isolated from Patients with Systemic Disease and from Healthy Carriers
SUMMARY: Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.
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- Physiology And Growth
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In Streptococcus faecium Penicillin-binding Protein 5 Alone Is Sufficient for Growth at Sub-maximal But Not at Maximal Rate
More LessSUMMARY: In Streptococcus faecium inhibition by both benzylpenicillin and cefotaxime of cells growing at maximal and at reduced rates was associated with saturation of different penicillin-binding proteins. Cells growing at reduced rates were not inhibited by benzylpenicillin concentrations that saturated all penicillin-binding proteins except penicillin-binding protein 5, but did stop growing when this protein was saturated.
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Concerted Induction of the S3 Alkylsulphatase of Pseudomonas C12B by Combinations of Alkyl Sulphates and Alcohols
More LessSUMMARY: Growth of the detergent-degrading bacterium Pseudomonas C12B on pyruvate supplemented with pure tetradecan-2-sulphate and tetradecan-2-ol together, led to induction of the S3 alkylsulphatase active towards symmetrical or near-symmetrical secondary alkyl sulphates. Induction was accompanied by disappearance of the tetradecan-2-sulphate surfactant and by the appearance of some of the S3 activity (up to 45% of the total) in the culture fluid. Induction did not occur when one or other or both of the C14 compounds was omitted from the minimal-pyruvate growth medium, nor with resting cell suspensions even with both compounds present. Provided that the pyruvate medium contained tetradecan-2-sulphate, good induction of S3 enzyme could also be achieved by replacing tetradecan-2-ol with any one of most secondary alcohols tested, including alkan-2-ols (C4 to C16), symmetrical alcohols (C5 to C13) and decanol isomers with the hydroxyl at C-3, C-4 or C-5. Primary alcohols were ineffective. Requirements for the ester component of the inducer combination were more closely defined; maintaining tetradecan-2-ol and pyruvate as constant components, no activity was detected with C3, C4 or C6 alkan-2-sulphates, very feeble activity with C8 and C10 homologues, and increasing activity from C12 to C16. Symmetrical and near-symmetrical alkyl sulphates (substrates of the S3 enzyme) and primary alkyl sulphates, all tested in combination with tetradecan-2-ol plus pyruvate, failed to induce significant amounts of S3 enzyme even at elevated concentrations. Critical micelle concentrations were measured for alkan-2-sulphates and correlated with co-inducer capacity and with the concentration dependence of induction by tetradecan-2-sulphate, which followed a hyperbolic saturation curve with Kco-inducer = 0.29 mm (inducer concentration giving half-maximum induction). Successful induction was also achieved with tetradecan-2-sulphate alone (no alcohol added), but only in the absence of pyruvate, under which circumstances hydrolysis of tetradecan-2-sulphate to the parent alcohol occurred as a prerequisite to bacterial growth. The collective results suggested that induction required not only a combination of secondary alcohol and long-chain alkan-2-sulphate in appropriate amounts (concerted induction), but also that their presence was required at a particular time, when the cells were actively growing and dividing.
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Mechanism of Dodecane Uptake by Whole Cells of Cladosporium resinae
More LessSUMMARY: Whole cells of Cladosporium resinae took up n-dodecane in two stages. The hydrocarbon was first passively adsorbed to the cell surface, and then hydrocarbon was taken in by a mechanism that obeyed Michaelis–Menten saturation kinetics [K m 1 mm, V max 12∙1 nmol min−1 (mg protein−1)]. Under conditions of poor agitation the initial adsorption limited uptake rates. The organism accumulated unaltered substrate to higher concentrations within the cytosol than in the surrounding medium, but this depended upon high concentrations of dodecane being adsorbed to the cell surface, thus creating a diffusion gradient.
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Regulation of Glycogen Synthase in Sclerotium rolfsii
R. SHAPIRA, M. PINES, I. CHET and Y. HENISSUMMARY: Changes in glycogen, extracellular polysaccharides, glycogen synthase (EC 2.4.1.11) and cAMP were followed during growth of submerged mycelium of Sclerotium rolfsii. While glucose was present in the medium glycogen and extracellular polysaccharides accumulated to maximum levels of 40 μg (mg dry wt)−1 and 0∙3 mg (mg dry wt)−1 respectively; they were degraded following glucose exhaustion. Neither glycogen nor extracellular polysaccharide concentrations were affected by increasing the glucose concentration in the growth medium from 0∙5 to 1∙5% (w/v). Following inoculation, both glucose-6-phosphate-dependent and glucose-6-phosphate-independent glycogen synthase activities increased during exponential growth, remained constant during the stationary phase as long as glucose was present in the medium, and declined rapidly after the exhaustion of glucose. The cAMP content of submerged cultures of S. rolfsii was 5 pmol (mg protein)−1; however, upon glucose exhaustion there was a sharp increase followed by a decrease to the initial basal level. The activity of glycogen synthase in crude extracts was increased by glucose 6-phosphate and inhibited by cAMP.
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Extracellular Pectinolytic Enzymes of Fungi Elicit Phytoalexin Accumulation in Carrot Suspension Culture
More LessSUMMARY: A heat-labile elicitor of phytoalexin accumulation in carrot (Daucus carota L.) was detected in culture filtrate of Chaetomium globosum grown in Czapek-Dox medium. The elicitor activity correlated with the activity of pectinolytic enzymes in the culture filtrates. The culture filtrates induced the release of heat-stable elicitors from carrot cell homogenates, pectic fraction of carrot cell walls and citrus pectin. The coincidence of heat sensitivity and sodium periodate inactivation profiles of elicitor and pectinolytic activity suggested that the activity of pectinolytic enzymes is essential for the release of the heat-stable elicitor. The culture filtrates of Botrytis cinerea, Fusarium moniliforme and Helminthosporium oryzae showed similar elicitation patterns.
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The Energetics and Kinetics of Extracellular Polysaccharide Production From Methanol by Micro-organisms Possessing Different Pathways of C1 Assimilation
More LessSUMMARY: Product excretion by Methylophilus sp. NCIB 12047, Pseudomonas extorquens NCIB 9399 and Pichia pastoris during growth on methanol was examined. These organisms possess the ribulose monophosphate pathway, the serine pathway and the dihydroxyacetone pathway of C1 assimilation, respectively. Only Methylophilus sp. NCIB 12047 produced significant amounts of extracellular product from methanol under conditions of nitrogen limitation in chemostat culture. This was a low-viscosity extracellular polysaccharide containing glucose and mannose in the ratio 3:1. Maximum polysaccharide production occurred under nitrogen limitation at a methanol/ammonium sulphate ratio > 10. The other two organisms responded to nitrogen limitation by increasing the rate of methanol oxidation to CO2. The maximum yield for polysaccharide production by Methylophilus sp. was 0·34 g (g oxygen)-1 and 0·30 g (g methanol)-1. The maximum specific rate of polysaccharide production was 0·18 g (g protein)-1 h-1. Methylophilus sp. grew readily under oxygen limitation and excreted an extracellular polysaccharide under these conditions. Examination of the biochemical pathways for polysaccharide production via the various C1 fixation routes indicates that the ribulose monophosphate pathway is energetically the most favourable. Polymer production by Methylophilus sp. is energetically neutral in terms of net ATP demand; however, the rate of ATP utilization for polymer production is equivalent to 65 to 80% of that required for cell production at the same growth rate. The results reported suggest that the energetic constraints imposed by the various pathways of C1 assimilation strongly influence both the rate of synthesis and the composition of exopolysaccharides produced by methylotrophs.
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Nitrogen Fixation by Gloeothece sp. PCC 6909: Respiration and Not Photosynthesis Supports Nitrogenase Activity in the Light
More LessSUMMARY: Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved O2 (dO2) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 μm-O2 depending on the age of the culture. At the optimum dO2, nitrogenase activity [typically 4 to 6 nmol C2H4 (mg protein)−1 min−1] was similar in the light or in the dark. Alteration of light intensity from zero to 2 klx, or addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), had no effect on nitrogenase activity. At 1 klx the ADP/ATP ratio was 0∙2 and showed only a marginal increase as the dO2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external dO2, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of dO2.
In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three O2-uptake systems with apparent K m values of 1 μm, 5 μm and 25 μm. The ADP/ATP ratio under anaerobic conditions was 0∙47 and showed a marked decrease as dO2 was increased to 25 μm. A CN-insensitive respiratory activity, which neither supported nitrogenase activity nor was coupled to ATP synthesis, was associated with the system with the apparent K m of 5 μm. The dependence of the specific activity of nitrogenase on dO2 indicated that both the high affinity (K m 1 μm) and low affinity (K m 25 μm) O2-uptake systems contributed ATP or reductant for N2-fixation. KCN (2∙5 mm) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and dO2. We conclude that respiration is the major source of reductant and ATP for nitrogenase activity both in the dark and in the light, but that photosystem I can contribute ATP at very high levels of illumination.
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Defining the Metabolic and Growth Responses of Porcine Haemophili to Exogenous Pyridine Nucleotides and Precursors
More LessSUMMARY: A variety of biologically important pyridine nucleotides and precursors were examined for their capacities to satisfy the V-factor requirement of 30 strains of porcine haemophili. Of the compounds tested, only NAD, NMN and nicotinamide riboside (NR) supported the growth of all strains; NADP supported the growth of only the type strain of Haemophilus parasuis. Further studies with the H. parasuis type strain and the neotype strain of H. pleuropneumoniae demonstrated that, during growth, these organisms exhibited affinities for NMN that were greater than those for NAD; the affinity of H. pleuropneumoniae for NR was similar to that for NMN, whereas H. parasuis exhibited relatively low affinity for NR. With either organism, equimolar amounts of NAD and NMN supported the production of approximately equal amounts of biomass whereas growth yields were substantially lower when NR was the pyridine nucleotide source. When either organism was grown in the presence of excess exogenous [carbonyl-14C]NAD, cessation of growth was accompanied by the apparent exhaustion of the NAD supply. Approximately 80% of the radioactivity added as [14C]NAD could be recovered as extracellular [14C]nicotinamide and the majority of the assimilated radioactive material was present intracellularly in the form of a [14C]NAD(P) pool. The results are discussed in terms of the structural features required of a pyridine compound for it to support the growth of porcine haemophili, the capacity of these organisms to compete for pyridine nucleotide sources in vivo, and possible mechanisms involved in the assimilation of such compounds.
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Mycobactin and the Competition for Iron between Mycobacterium neoaurum and M. vaccae
More LessSUMMARY: Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0·02–0·05 μg Fe ml-1) and iron-sufficiency (2–4 μg Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6–8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells.
Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.
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Iron Uptake Processes in Mycobacterium vaccae R877R, a Mycobacterium Lacking Mycobactin
More LessSUMMARY: The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.
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- Systematics
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Characterization of Haemophilus parainfluenzae Strains with Low-M r or Ladder-like Lipopolysaccharides
More LessSUMMARY: Twenty-five Haemophilus parainfluenzae strains were characterized for lipopolysaccharide (LPS) profiles, outer membrane protein profiles, serum sensitivity, plasmid profiles and DNA homology. Seventeen strains produced low-M r LPS that did not contain O-sidechains, while the remaining eight strains contained ladder-like LPS suggestive of O-repeated units. This is the first time in the genus Haemophilus that LPS with O-repeated groups has been described. The strains producing the different types of LPS could not be distinguished from each other in outer membrane protein profiles or the other characteristics examined.
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Distribution and Application of Mycobactins for the Characterization of Species within the Genus Rhodococcus
More LessSUMMARY: Representatives of 11 species of Rhodococcus were examined for their ability to synthesize mycobactin, a lipid-soluble siderophore, following iron-limited growth on solidified glycerol/asparagine medium. Rhodococcus bronchialis, R. terrae and R. rubropertinctus formed mycobactins, whereas the remaining species (R. coprophilus, R. equi, R. erythropolis, R. rhodnii, R. rhodochrous, R. ruber, R. maris and R. luteus) failed to synthesize these compounds even under conditions of strictly iron-limited growth. The mycobactins from R. terrae and R. rubropertinctus showed close similarity by thin-layer chromatography and high-performance liquid chromatography and could be easily distinguished from that of R. bronchialis.
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