- Volume 138, Issue 10, 1992
Volume 138, Issue 10, 1992
- Physiology And Growth
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Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ratios
More LessNADH/NAD+ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ratios.
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Physiological analysis of mutants of Saccharomyces cerevisiae impaired in sulphate assimilation
More LessThe assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes. To date, the functions of nine of these genes are still not clearly established. A set of strains, each bearing a mutation in one MET gene, was studied. Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase. These genes are MET1, MET5, MET8, MET10 and MET20. Wild-type strains of S. cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources. They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate. Here we show that both of the two sulphur atoms of thiosulphate are used by S. cerevisiae. Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.
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The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae
More LessSaccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells. The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid. Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells. The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa. In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble). In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose. 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium. These results suggest that in S. cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the ‘Ehrlich Pathway’.
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Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis
More LessBacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 °C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.
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Cell surface characteristics of Lactococcus lactis harbouring pCI528, a 46 kb plasmid encoding inhibition of bacteriophage adsorption
More LesspCI528 is a 46 kb plasmid, originally isolated from Lactococcus lactis subsp. cremoris UC503, which causes a decrease in bacteriophage adsorption in lactococcal strains. Cell surface characteristics of two pCI528-containing strains, L. lactis subsp. cremoris UC503 and L. lactis subsp. lactis UC505, were compared with those of their isogenic pCI528-cured derivatives, UC563 and MG1363, respectively. Following centrifugation, strains containing pCI528 produced a loose fluffy pellet. The presence of pCI528 also caused a ‘hard’ colony morphology on agar media. The fluffy pellet effect mediated by pCI528 could be eliminated by washing cells in 0.05 M-NaOH, which also restored full sensitivity to phage in the L. lactis subsp. lactis UC505 background. GLC analysis of cell wall material indicated that pCI528-containing hosts had significantly elevated levels of both galactose and rhamnose. Marked alterations in the cell surface of strains harbouring pCI528 were observed in electron micrographs.
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Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a β-lactamase fusion protein
More LessA secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to β-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-β-lactamase fusion protein was processed much more slowly than the fusion of β-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.
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Characterization of an anaerobic fungus from Ilama faeces
More LessAn anaerobic fungus was isolated from Ilama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 °C with an optimum of around 38 °C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal ‘globules’ and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia ‘crystal bodies’ and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.
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- Plant-Microbe Interactions
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Role of calcium in adhesion and germination of zoospore cysts of Pythium: a model to explain infection of host plants
More LessNewly-formed zoospore cysts of Pythium aphanidermatum adhered to glass and germinated without a further trigger, but both adhesion and germination were suppressed by EGTA. Older (10 min) cysts adhered and germinated poorly unless supplied with Ca2+. Adhesion and germination were linked but separable, because young cysts germinated when dislodged from glass after 10 min, and older cysts kept in suspension germinated in response to high (7–10 mM) concentrations of Ca2+, Mg2+or Sr2+. Other cations (Li+, Na+, K+, Ba2+, Mn2+, Fe3+, Cu3+) had no effect up to 25 mM concentration or were toxic. The naturally low germination of suspended cysts was suppressed by EGTA (chelator), La3+or verapamil (Ca2+entry blockers), amiloride (Ca2+flux inhibitor), trifluoperazine or dibucaine (calmodulin antagonists), TMB-8 (intracellular Ca2+antagonist) or A23187 (Ca2+ionophore). Suppression by TMB-8 or A23187 was not relieved by any post-treatment. Suppression by EGTA was relieved only by divalent cations. Suppression by all other compounds was relieved only by specific L-amino acids that stimulated germination of control cysts (asparagine, aspartic acid and glutamic acid but not lysine or glutamine, where tested). Alanine relieved suppression by verapamil or dibucaine in Pythium dissotocum but not P. aphanidermatum. The findings indicated a central role of Ca2+(presumably released during encystment) in adhesion and germination, such that zoospores would germinate autonomously by a Ca2+-mediated cascade when they dock and encyst on roots with the fixed point of germination adjacent to the host. Amino acids are not essential but apparently synergize this process by species-specific receptor-binding which facilitates Ca2+uptake or Ca2+signalling.
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Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep
More LessLipopolysaccharide (LPS) from 40 isolates of Pasteurella haemolytica, comprising 23 serotype A1, seven serotype A2, one serotype T4, one serotype T10 and eight untypable isolates, obtained from diseased and healthy cattle or sheep, was characterized by SDS-PAGE and Western blotting. Ten different SDS-PAGE LPS profiles, five smooth and five rough, were identified among the biotype A and untypable isolates and designated LPS types 1–10. LPS types 1 and 2 were smooth, had similar O-antigen banding-patterns but differed in the low-molecular-mass or core-oligosaccharide regions; type 3 LPS was rough but had a core-oligosaccharide region similar to that of LPS type 1. No similarities were observed between these LPS types and types 6, 7 and 9, which were smooth, and types 4, 5, 8 and 10, which were rough. Most serotype A1 isolates (19/23) were of LPS type 1, whereas two isolates each had LPS of types 2 and 3. The majority (5/7) of serotype A2 isolates possessed type 3 LPS, whereas the remaining two isolates each had LPS of types 4 and 5. There was much greater heterogeneity within the untypable group of isolates, which comprised LPS of types 1 and 9 (two isolates each), and 6, 7, 8 or 10 (one isolate each). Western blotting analysis demonstrated that LPS types 1 and 2 had immunologically identical O-antigen side-chains but differed in their core-oligosaccharide regions, whereas the core-oligosaccharide region of rough LPS type 3 was immunologically very similar to that of LPS type 1. The other LPS types were immunologically unrelated to these three LPS types. The majority (20/23) of serotype A1 isolates originated from cattle and possessed LPS types 1 or 2, different from most (5/7) of the serotype A2 isolates which originated from sheep and possessed LPS of types 3 or 4. However, two of the three ovine serotype A1 isolates had the same type 3 LPS as occurred in most of the ovine serotype A2 isolates, suggesting a possible correlation between LPS type and host specificity. This study has demonstrated that LPS diversity within different serotypes of P. haemolytica is greater than was previously thought and that certain LPS types might be host-specific.
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Targeting of specific domains of diphtheria toxin by site-directed antibodies
More LessAntibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141–157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224–237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513–526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominately against a narrow region within the peptide, consisting of only 5–6 aa residues. The predicted location of the peptide and their epitopes was confirmed by inspection of the X-ray crystallographic structure of DTx. Antibodies to peptides were selective for the toxin, one binding to DTx some 5–60-fold better than to diphtheria toxoid, presumably reflecting variability caused by toxoid preparation at this epitope. None of the antisera produced protected against DTx challenge in the guinea pig intradermal test in vivo. Although the availability of site-specific antibodies that recognize neutralizing epitopes would be very valuable, antibodies such as those described here should prove extremely useful in the structure-function analysis of DTx.
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