- Volume 136, Issue 7, 1990
Volume 136, Issue 7, 1990
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Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus
More LessA gene of Streptococcus sobrinus 6715 (serotype g ) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.
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Transformation of vegetative cells of Bacillus anthracis with plasmid DNA
More LessMethods have been developed for chemical transformation and electro-transformation (electroporation) of vegetative cells of Bacillus anthracis with supercoiled plasmid DNA. Chemical transformation was dependent on incubation in Tris/HCl with osmotic support and transformation with plasmid DNA was effected by treatment with polyethylene glycol 3350. Maximum transformation frequencies were 3·8 × 10−5 transformant c.f.u. per viable c.f.u. (1 × 103 c.f.u. per g DNA). Optimal frequencies were pH dependent and were affected by growth-medium composition. Transformation was not observed with linear or multimeric plasmid DNA. Electro-transformation of B. anthracis using high field intensity electroporation was dependent on the composition of both the growth medium and the electroporation buffer. Maximum electro-transformation frequencies were 5·3 × 10−4 c.f.u. per viable c.f.u. (2.6 × 104 c.f.u. per g DNA). The use of early exponential phase cells was critical to both procedures and the maximum efficiency (c.f.u. per g DNA) of each system was strain dependent under the conditions described.
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Induction and catabolite repression of high-affinity glucoamylase in Aspergillus terreus strain 4
More LessThe regulatory mechanism for glucoamylase synthesis in Aspergillus terreus strain 4 was studied. Synthesis of the enzyme was induced in the presence of maltose and other carbohydrates containing maltose units. Smaller amounts of enzyme were produced in the presence of glucose or other monosaccharides like sorbose and xylose. Enzyme production was maximal with 0·5% starch as the carbohydrate source. At higher concentrations of starch, production of the enzyme was less. In glucose medium, both extracellular and intracellular glucoamylase activities were very low. When starch medium was supplemented with glucose, the extracellular enzyme activity decreased with increasing concentration of added glucose. Intracellular amylase was also detected. There was no appreciable change in the level of this enzyme activity in the presence of added glucose. Further, when glucose was added to the growing culture after 2 d in starch medium there was a marked decrease in glucoamylase synthesis, which was also proportional to the amount of glucose added. Enzyme synthesis was resumed as soon as the glucose concentration in the culture medium fell below a critical level. All these observations strongly suggest that in A. terreus strain 4, extracellular glucoamylase synthesis is subject to catabolite repression.
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Pathways of arginine biosynthesis in extreme thermophilic archaeo-and eubacteria
More LessThe pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus aquaticus and Thermotoga maritima, and in two thermophilic archaeobacteria, Sulfolobus solfataricus and Pyrococcus furiosus. In the first three organisms, arginine biosynthesis proceeds via N-acetylated intermediates as in mesophilic microorganisms. Only the enzymes catalysing the three last steps of the pathway could be detected in P. furiosus. The two eubacterial strains possess an ornithine acetyltransferase and are thus able to recycle the acetyl group from acetylornithine to glutamate. The archaeobacterium, S. solfataricus, uses the linear pathway in which the formation of ornithine is mediated by the hydrolytic enzyme acetylornithinase. Repression of enzyme synthesis by arginine was observed for most of the enzymes tested in T. aquaticus and S. solfataricus. Feedback inhibition by arginine was shown only on the ornithine acetyltransferase from T. aquaticus. This inhibition pattern is of interest since it would be the first example of control of arginine biosynthesis at this particular step. Data concerning the thermal stability of the arginine biosynthetic enzymes are presented.
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β-Glucosidase and cellulase formation by a Trichoderma reesei mutant defective in constitutive β-glucosidase formation
More LessA mutant of Trichoderma reesei QM 9414 - T. reesei M8 - was isolated after γ-irradiation. It did not form β-glucosidase during growth on glycerol or glucose, but secreted β-glucosidase upon growth on cellobiose. The mutant was also able to grow on cellulose and secrete β-glucosidase, but with a longer lag. β-Glucosidase activity could be induced in the mutant in a replacement medium by cellobiose and β-methyl d-glucoside. Mycelia lacking β-glucosidase were able to take up both these inducers immediately, indicating the presence of a constitutive permease. The mutant produced cellulases upon growth on lactose and - after a lag - on cellulose. Mycelia of T. reesei M8 pregrown on glycerol or cellobiose could be induced by sophorose to produce cellobiohydrolase I in a replacement system. The findings show that (i) cellobiose and other β-linked disaccharides can be taken up by T. reesei without prior hydrolysis, and (ii) that cellobiose most probably induces β-glucosidase formation during growth on cellulose.
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Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206
More LessGrowth of the yeast Pichia pastoris IFP 206 in methanol- and glucose-containing media led respectively to very and weakly flocculent cells. Mannans from both kinds of cells were extracted and compared. Chemical analysis and molecular mass estimation showed some differences between the mannans from very and weakly flocculent cells, especially in quantitative amino acid content. 1H NMR analysis showed that both kinds of mannan contained α-1,2 and β-1,2 linkages. Two acetolysis conditions, combined with 1H NMR analysis, revealed that mannans from both kinds of cells were composed of mannose, mannobiose, mannotriose, mannotetraose and mannopentaose side-chains with the following respective structures: Man; Manα1→2Man; Manα1→2Manα1→2Man; Manβ1→2Manα1→2Man; Manβ1→2Manβ1→2Manα1→2Man; Manα1→2Manβ1→2Manβ1→2Manα1→2Man. Additionally the β-1,2 linkages of the non-reducing terminal residues of the mannotetraose were shown to be acetolysislabile. The mannans from very flocculent cells were richer in mannopentaose than the mannans from weakly flocculent cells. According to these results, the extended conformations in the branching moieties of the mannan could be the basis of the higher degree of flocculation of the methanol-grown cells.
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Conversion of methionine to phytotoxic 3-methylthiopropionic acid by Xanthomonas campestris pv. manihotis
E. Ewbank and H. MaraiteIncubation of the plant pathogenic bacterium Xanthomonas campestris pv. manihotis with either l-[3,4-14C]methionine or [3,4-14C]KMBA (2-keto-4-methylthiobutyric acid) led to the production of [14C]MTPA (3-methylthiopropionic acid) and [14C]MTAA (3-methylthioacrylic acid). When an excess of non-radioactive KMBA was present in the medium, formation of [14C]KMBA from l-[3,4-14C]methionine was observed. Conversely, [14C]methionine accumulated in a trapping pool of non-radioactive methionine as a result of biotransformation of [3,4-14C]KMBA. Aminooxyacetic acid, a transaminase inhibitor, suppressed totally the formation of [14C]MTPA from l-[3,4-14C]methionine but not from [3,4-14C]KMBA. Metabolism of l-[1-14C]methionine liberated 14CO2 but did not produce [14C]MTPA. These results demonstrate that methionine is catabolized by X. campestris pv. manihotis into MTPA via transamination and subsequent decarboxylation of the intermediate a-keto acid KMBA.
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Growth of Bacillus stearothermophilus on glycerol in chemostat culture: expression of an unusual phenotype
More LessBacillus stearothermophilus grew readily on glycerol in carbon-limited chemostat culture and expressed a high carbon conversion efficiency. However, the strain of organism used (probably B. stearothermophilus var. nondiastaticus) proved particularly sensitive to glycerol, both respiration and growth being severely impeded by any surfeit of this compound. Sensitivity was found to correlate with an exceptionally high level of expression of glycerol kinase [activities of more than 80 μmol min−1 (mg protein)−1 were manifest in crude cell-free extracts], coupled with low activities of methylglyoxal synthase and of glyoxylase (enzymes of the methylglyoxal bypass). It is proposed that metabolic dysfunction results from an uncontrolled gross accumulation of glycerol phosphate (and early products of its metabolism) within the cells, coupled with depletion of the intracellular phosphate pool.
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Glycine-induced cryotransformation of plasmids into Bacillus anthracis
More LessDifferent cloning vectors (pC194, pBC16, pUB110, pBD10, pBD8, pAMβ1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about 103 c.f.u. per μg DNA) and allows different cloning vectors with molecular masses ranging from 1·8 to 17·7 MDa to be introduced into B. anthracis.
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Nucleotide sequences of the Bacillus subtilis flaD locus and a B. licheniformis homologue affecting the autolysin level and flagellation
More LessA DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced. The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene. Unexpectedly, the sequence of the flaD locus was identical to that of sin [sporulation inhibition gene; Gaur, N. K., Dubnau, E. & Smith, I. (1986). Journal of Bacteriology 168, 860–869]. A B. licheniformis homologue (flaL) of the B. subtilis flaD locus was cloned into pUC19 and identified by colony hybridization. The B. licheniformis DNA was subcloned and sequenced. Two ORFs (ORF1, or L-ORF1; and ORF2, orflaL) were detected, encoding 58 and 111 amino acid residues, respectively. These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B. subtilis DNA. The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those offlaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively. The regions 3′ of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription. The characteristics of the amino acid sequences are also discussed.
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Nutritional stress proteins in Candida albicans
More LessStarving cells of Candida albicans synthesize at least seven proteins that represent nutritional-stress proteins (NSP). Such NSPs are formed by both germination-competent and germination-deficient strains of C. albicans. Heat-shock proteins (HSP) are not formed by starving cells. Germination-competent cells synthesize specific sets of proteins when incubated in a starvation medium that contains the germ-tube-inducing substances N-acetyl-d-glucosamine or l-proline. Both sets of induced proteins were also synthesized by a germination-deficient strain of C. albicans.
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The cellulase complex of Neurospora crassa: activity, stability and release
More LessThe temperature and pH optima, and the temperature and pH stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus Neurospora crassa were investigated. The effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of the cellulase complex were also examined. For the different enzymes of the complex, activity maxima occurred between pH 4·0 and 7·0, with pH 5·0 being close to optimal for stability of all. Temperature optima for activity ranged between 45 and 65 °C, with the stability optimum between 45 and 50 °C. The presence of C18 fatty acids and surfactants resulted in increased production of both endoglucanase and exoglucanase in the medium. Oleic acid was the most effective fatty acid tested, and Tween 80 the most effective surfactant. Oleic acid had no detectable effect on production of β-glucosidase, and Tween 80 actually reduced its production.
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Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species
More LessA collection of 117 strains of Haemophilus influenzae, including 112 non-typable isolates recovered predominantly in the USA and France from genital, obstetric and neonatal sources, was characterized by the electrophoretic mobilities of 10 metabolic enzymes. Eighty-six distinctive multilocus chromosomal genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Isolates of five allied biotype IV ETs were highly divergent from all other strains and hybridization of chromosomal DNA revealed that they undoubtedly represent a previously unrecognized species of Haemophilus. Isolates representing these ETs were recovered predominantly from obstetric infections and serious neonatal diseases and apparently possess specific tropism for the genital tract. Strains of these five ETs were present in samples from both the USA and France, but only in the USA did they cause bacteraemia and meningitis, an occurrence which probably reflects differences in patient management between the two countries. Although strains assigned to H. influenzae (sensu stricto) were strongly polymorphic in multilocus enzyme genotype, 69% of isolates recovered from patients with meningitis and/or septicaemia were assigned to only two clone families, a result suggesting that some serologically non-typable strains of H. influenzae originating from the genital tract are unusually virulent.
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Effect of ethanol on the phospholipid and fatty acid content of Schizosaccharomyces pombe membranes
More LessEthanol at concentrations up to 5% (v/v) had no effect on the growth of Schizosaccharomyces pombe, whereas concentrations over 7.5% were inhibitory. The major membrane phospholipids in S. pombe cells growing aerobically in the absence of added ethanol were phosphatidylinositol, phosphatidylcholine and phosphatidyl-ethanolamine. Oleic acid (18:1) was the main fatty acid. When ethanol (7.5%) was added to aerobically growing cultures, the phosphatidylinositol content increased, whereas the 18:1 content decreased. Similar changes were observed in the membrane phospholipids of cells grown anaerobically without ethanol. However, the presence of ethanol in anaerobically growing cultures had an opposite effect on fatty acids, as the 18:1 content increased. The results support the idea that ethanol tolerance in S. pombe may be connected with a high content of 18:1 fatty acids, and with the ability to maintain a high rate of phospholipid biosynthesis.
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IncP-mediated transfer of loci involved with gas vesicle production in Ancylobacter aquaticus
More LessIncP plasmids were transferred by conjugation from Escherichia coli to gas-vacuolate strains of Ancylobacter aquaticus, between strains of A. aquaticus, and from A. aquaticus to E. coli. These plasmids would also mobilize the chromosome of A. aquaticus. In this way, A. aquaticus genes could complement mutations in either A. aquaticus or E. coli. A locus involved in gas vesicle production was co-transferred with rifampicin resistance in matings between strains of A. aquaticus.
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Molecular cloning and nucleotide sequence of a gene for alkaline celluase from Bacillus sp. KSM-635
More LessA gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2·6 kb and 4·0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2·4 kb region which was indispensable for the production of cellulase, and the flanking, 1·1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a σ 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.
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Alterations in nucleotide and pyrophosphate levels in Phytophthora palmivora following exposure to the antifungal agent potassium phosphonate (phosphite)
More LessShort exposure (up to 3 h) of phosphate-starved mycelium from Phytophthora palmivora to the antifungal agent potassium phosphonate resulted in decreased levels of NAD, ATP, and a number of compounds tentatively identified as polyphosphorylated nucleotides (alarmones). ADP, AMP and adenosine levels were not increased, as would be expected if phosphorylation were the site of inhibition. Pyrophosphate levels, however, were raised. The incorporation of [32P]phosphate into phospholipids and other macromolecules was not affected during this short exposure. Together, these results suggest that adenylate synthesis may be a primary site of action of phosphonate in the fungus.
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