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Volume 140,
Issue 6,
1994
Volume 140, Issue 6, 1994
- Pathogenicity And Medical Microbiology
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Isolation and characterization of the haeminbinding proteins from Neisseria meningitidis
More LessThe mechanism of haem-iron acquisition in Neisseria meningitidis is poorly understood. Using haemin-agarose in a batch affinity chromatography method, two haemin-binding proteins of 97 and 50 kDa were isolated from total membranes derived from Neisseria meningitidis B16B6 grown under iron-deficient but not under iron-replete conditions. No binding proteins were affinity-purified when total membranes underwent limited proteolysis with trypsin, suggesting a haem-protein interaction. When biotinylated human haemoglobin was used as the affinity ligand, proteins of identical molecular mass were isolated. Detection of haemin-binding proteins in a whole cell binding assay demonstrated a surface-exposed location. Competitive binding studies indicated that this haem-protein interaction was specific, because only haemin or human haemoglobin, but not cytochrome c111' protoporphyrin IX, iron-loaded human lactoferrin, iron-loaded human transferrin or Fe(NO3)3, could abrogate binding. The presence of similar haemin-binding proteins in a limited survey of clinical meningococcal strains indicated that the expression of the haemin-binding proteins is not serogroup-specific.
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- Physiology And Growth
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Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase
More LessAzotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o- and bd-types. The latter is required for respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities (K m) for oxygen uptake by A. vinelandii cells and membranes respiring dl-malate have been determined by using the deoxygenation of oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen concentration. Dual-wavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0·003–10 μM, and revealed three distinct affinities for oxygen in a wild-type strain. The kinetic properties of each oxidase were distinguished by the use of two mutants, one lacking and one over-producing the cytochrome bd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high affinity oxidase in all three strains with K m values for membrane preparations of 0·013–0·019 μM. In strains having the cytochrome bd-type oxidase, the K m values measured with intact cells were approximately fourfold higher than in membranes. These results suggest a barrier to the transfer of oxygen to the high affinity component by cytochrome bd, perhaps due to very fast oxygen binding or scavenging by cytochrome d, or to the location of the oxygen-consuming sites of these oxidases on different faces of the membrane. The deoxygenation kinetics of oxymyoglobin revealed the presence of two components with mean K m values of about 0·33 and 4·5 μM. The 4·5 μM component is attributed to the cytochrome bd-type oxidase because it was lacking in intact cells and membranes of the cytochrome bd-deficient mutant strain. The other two components (one with a mean K m value of about 0·33 μM and the highest affinity activity) could not be assigned to particular oxidase(s). The results are interpreted in relation to the physiological role of the cytochrome bd-terminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome bd-type oxidase in other bacteria.
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A spore-lytic enzyme released from Bacillus cereus spores during germination
More LessThe exudate of fully germinated spores of Bacillus cereus IFO 13597 in 0·25 M sodium phosphate buffer, pH 7·0, was found to contain a spore-lytic enzyme. This enzyme was found to cause loss of absorbance in coat-stripped spore suspensions and phase-darkening of the spores but had minimal activity on isolated peptidoglycan substrates. The enzyme was purified in an active form and identified as a 24 kDa protein which is either an amidase or a peptidase. The amino-terminal 19 residues had the following sequence: FSNQVIQRGASGEKVIELQ. The spore-lytic enzyme retained its activity in a medium of a relatively high ionic strength containing a non-ionic surfactant such as nonaethyleneglycol n-dodecyl ether. This activity was optimum at a salt concentration of about 30 mM in assay buffer at neutral pH. In contrast to the enzyme in a spore-bound form, the enzyme in solution was shown to be heat-sensitive and was readily inactivated by thiol reagents.
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Effect of growth arrest on carotene accumulation and photosynthesis in Dunaliella
More LessThe halotolerant green alga Dunaliella bardawil is known to accumulate β-carotene in response to stress factors such as high light intensity, high salt concentrations and nutrient limitation. In this report, the accumulation of β-carotene was studied in cells from nitrate-limited chemostat cultures, in comparison with those of D. salina, a strain that does not accumulate β-carotene under stress conditions. D. bardawil responded to growth arrest by accumulating β-carotene and, to a lesser degree, lutein and zeaxanthin. A substantial fraction of β-carotene and all the lutein and zeaxanthin was associated with the thylakoid fraction. The accumulation of carotenoids in D. bardawil occurred only in the light, but the light intensities were far below those where the photosynthetic rate is maximal. After growth arrest, the amount of chlorophyll (Chi) decreased in both strains. However, in D. bardawil Chi a decreased to a lesser extent in comparison with Chi b, which resulted in an increased Chi alb ratio. The maximum photosynthetic capacity declined rapidly in both strains after growth arrest. In contrast, the photosynthetic efficiency showed a temporary increase in D. bardawil and a decrease in D. salina. This increase did not occur when carotenogenesis was inhibited by diphenylamine, implying a causal relationship between enhanced carotenogenesis and increase of photosynthetic efficiency. The possible involvement of stress-accumulated carotenoids in photosynthetic activity is discussed.
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Methanesulphonate utilization by a novel methylotrophic bacterium involves an unusual monooxygenase
More LessMethylotroph strain M2, isolated from soil, was capable of growth on methanesulphonic acid (MSA) as sole carbon and energy source. MSA was oxidized by cell suspensions with an MSA: oxygen stoichiometry of 1·0:2·0, indicating complete conversion to carbon dioxide and sulphate. The presence of formaldehyde and formate dehydrogenases and hydroxypyruvate reductase in MSA-grown bacteria indicated the production of formaldehyde from MSA (and its further oxidation for energy generation), and assimilation of formaldehyde by means of the serine pathway. Growth yields in MSA-limited chemostat culture were a function of dilution rate, with yield ranging from 7·0 g mol-1 at D = 0·04 h-1, to 14·6 at 0·09 h-1. MSA metabolism was not initiated by hydrolysis to produce either methane or methanol, but appears to be by an NADH-dependent methanesulphonate monooxygenase, cleaving MSA into formaldehyde and sulphite. The organism lacked ribulose bisphosphate carboxylase and did not fix carbon dioxide autotrophically. It also lacked ribulose-monophosphate-dependent hexulose phosphate synthase. Growth on methanol, methylammonium and other C1 compounds was exhibited, but ability to oxidize MSA was not induced by growth on these substrates. Similarly, methylammonium (MMA) was only oxidized by strain M2 grown on MMA. Growth on methanol involved a pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase (large subunit molecular mass 60 kDa). This organism is the first methylotroph shown to have the ability to oxidize MSA, by virtue of a novel monooxygenase, and is significant in the global sulphur cycle as MSA can be a major product of the oxidation in the atmosphere of dimethyl sulphide, the principal biogeochemical sulphur gas.
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- Plant-Microbe Interactions
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An exoB mutant of Rhizobium sp. is effective in indeterminate nodules of Hedysarum coronarium
A Rhizobium sp. (Hedysarum coronarium) calcofluor dark (Cal-) mutant, named Cal10, was obtained following Tn5mob-insertion mutagenesis. It is affected in the synthesis of exopolysaccharide and presents an altered lipopolysaccharide that is not recognized by a polyclonal antibody against the lipopolysaccharide of the parental strain. The residual exopolysaccharide obtained from the mutant lacks galactose and the high-molecular-mass acidic fraction. This mutant was complemented by plasmid pD56 that restores the production of exopolysaccharide, the alteration of lipopolysaccharide and the Cal phenotype. The data presented indicate that the gene in which the mutant is defective is homologous to the exoB gene of Rhizobium meliloti and fails to synthesize UDP-glucose 4′-epimerase. The Cal10 mutant was Fix+ on H. coronarium (sulla) although it develops an indeterminate type of nodule, indicating that exopolysaccharide is not essential for a successful nodulation in this symbiotic association.
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- Systematics
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Evidence for a phylogenetic connection between Coccidioides immitis and Uncinocarpus reesii (Onygenaceae)
More LessCoccidioides immitis is an anomaly amongst the human systemic fungal pathogens. Its unique parasitic cycle has contributed to confusion over its taxonomy. Early investigators mistakenly suggested that the pathogen is a protist, while others agreed it to be a fungus but placed it in four different divisions of the Eumycota. The taxonomy of C. immitis is still unresolved. Ultrastructural examinations of its parasitic and saprobic phases have revealed features that are diagnostic of the ascomycetous fungi. Moreover, striking similarities between the kind of asexual reproduction (i.e. arthroconidium formation) of this pathogen and certain anamorphic and teleomorphic members of the genus Malbranchea have suggested a close relationship. Teleomorphs of these Malbranchea species are members of the Onygenaceae (Order, Onygenales). This family also includes teleomorphs of two human respiratory pathogens, Histoplasma capsulatum and Blastomyces dermatitidis. Although the 18S rRNA gene sequences (1713 bp) of these two pathogenic forms differ from that of C. immitis by only 35 and 33 substitutions, respectively, their mode of conidiogenesis is characterized by production of solitary aleurioconidia rather than alternate arthroconidia. In this study we have used characters derived from biochemical, immunological and molecular analyses to compare relatedness between C. immitis, H. capsulatum, B. dermatitidis, and six non-pathogenic species of Malbranchea (the Malbranchea states of Uncinocarpus reesii and Auxarthron zuffianum, as well as M. albolutea, M. dendritica, M. filamentosa and M. gypsea). Evidence is presented which supports inclusion of C. immitis in the Onygenaceae, and indicates that a close phylogenetic relationship exists between the Malbranchea state of U. reesii and this respiratory pathogen.
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Isozyme analysis of anaerobic rumen fungi and their relationship to aerobic chytrids
More LessIsozymes of 23 cultures of the anaerobic rumen fungi and seven cultures of aerobic chytridiomycete fungi were analysed by PAGE. A total of 14 isozyme loci were successfully typed by PAGE. They were peptidase A & C-1, peptidase A & C-2, peptidase D-1, peptidase D-2, malate dehydrogenase-1, malate dehydrogenase-2, esterase-1, esterase-2, malic enzyme-1, malic enzyme-2, isocitrate dehydrogenase, shikimate dehydrogenase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. Isozyme analysis can be used for studying the genetic relationships among the different anaerobic rumen fungi and the aerobic chytridiomycete fungi and the isozyme characteristics can serve as additional taxonomic criteria in the classification of the anaerobic rumen fungi. A dendrogram based on the isozyme data demonstrated that the anaerobic rumen fungi formed a cluster, indicating a monophyletic group, distinctly separated from the aerobic chytridiomycete fungi. Piromyces communis and P. minutus showed a close relationship but P. spiralis showed a more distant relationship to both P. communis and P. minutus. Piromyces as a whole was more related to Caecomyces than to Neocallimastix. Orpinomyces was also found to be more related to Piromyces and Caecomyces than to Neocallimastix. Orpinomyces intercalaris C 70 from cattle showed large genetic variation from O. joyonii, indicating that it is a different species.
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- Genome Analysis
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Characterization of a Streptomyces-lividans-type site-specific DNA modification system in the avermectin-producer Streptomyces avermitilis permits investigation of two novel giant linear plasmids, pSA1 and pSA2
More LessThe degradation of Streptomyces avermitilis DNA samples analysed by conventional pulsed-field gel electrophoresis was shown to be due to Trisdependent, double-strand cleavage. Using alternative electrophoretic conditions, separation of intact DNA molecules was achieved, permitting the identification of two novel giant linear plasmids: the 100 kb pSA1 and 250 kb pSA2. Use of pSA2 DNA as a probe showed that pSA1 does not cross-hybridize indicating that the plasmids are not closely related. The site-specificity of the DNA modifications, which render the DNA susceptible to Tris-dependent cleavage, was found to be essentially identical to that of similar modifications found in the DNA of S. lividans.
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Electrophoretic karyotype of Fusarium solani
More LessThe electrophoretic karyotype of Fusarium solani was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Thirteen chromosomal bands were obtained. The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe and Saccharomyces cerevisiae, is estimated to be between 0·42 and 6·08 Mb, the total molecular size of the DNA being 39·90 Mb.
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