- Volume 136, Issue 7, 1990
Volume 136, Issue 7, 1990
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Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224
More LessA conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.
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Discovery of an insertion sequence, IS116, from Streptomyces clavuligerus and its relatedness to other transposable elements from actinomycetes
We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1·4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and - most particularly - IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.
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Investigation of mutants of Methylophilus methylotrophus which are defective in methanol oxidation
More LessFive classes of mutants of Methylophilus methylotrophus which are defective in methanol oxidation (Mox-) have been isolated, partially characterized, and complemented using a Sau3A genomic library. Two classes of mutants were defective in production of the a subunit of methanol dehydrogenase, another two classes in the production of both the a subunit and cytochrome c L, while the fifth class produced the a subunit, but did not contain an active methanol dehydrogenase, nor cytochrome c L. The genes defective in these mutants were linked.
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Calcium deficiencies and apical hyperbranching in wild-type and the ‘frost’ and ‘spray’ morphological mutants of Neurospora crassa
More LessThe role of Ca2+ in the maintenance of apical dominance in Neurospora crassa was investigated. In the presence of the calcium-channel blocker verapamil (1 mm), wild-type hyphal tips demonstrated enhanced branching which led to a fan-like pattern of growth, similar to that seen in certain of the morphological mutants of N. crassa such as ‘frost’ and ‘spray’. In verapamil-treated hyphae, unlike untreated controls, Ca2+ was not observed in hyphal tips by fluorescence microscopy and the exaggerated branching pattern could be corrected by the addition of 10 mm-Ca2+. Studies using the morphological mutants ‘frost’ and ‘spray’, which grow typically on minimal medium with a branching pattern quite similar to verapamil-treated wild-type, also failed to demonstrate Ca2+ in hyphal tips. Exogenously added Ca2+ (50–500 mm) almost completely corrected the abnormal branching seen in these mutants, converting them to an essentially wild-type appearance. These observations suggest that low Ca2+ levels, induced in the wild-type by verapamil, and constitutive in the mutants, are responsible for the abnormal branching patterns.
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Inducible UV repair potential of Pseudomonas aeruginosa PAO
More LessPseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.
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Effect of ethanol on cell growth, metabolism and cation fluxes in the yeast Metschnikowia reukaufii
More LessThe yeast Metschnikowia reukaufii could not utilize ethanol as a growth substrate, although ethanol stimulated cellular O2 consumption and the reduction of intracellular nicotinamide adenine nucleotides. Also, ethanol inhibited cell growth in glucose-containing medium. The effect was stronger when the K+ concentration in the growth medium was lowered from 5.8 to 0.6 mM. Addition of glucose to an aerated cell suspension caused an initial H+ efflux and K+ influx in a ratio of approximately 1:1. This was followed by a phase of continuing extracellular acidification without any measurable uptake of K+. In contrast, in cells energized by glucose, ethanol stimulated K+ efflux; concomitantly, H+ extrusion was markedly lowered by ethanol. The rates of H+ extrusion correlated with the intracellular level of glucose 6-phosphate and not of ATP. It is concluded that there is a regulatory interaction, though not by a direct effect, between glucose 6-phosphate and the plasma-membrane ATPase. Ethanol appears to activate electron transfer from cytosolic NADH to O2 by a pathway independent of the mitochondrial respiratory chain.
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Characterization of a glycerol kinase mutant of Aspergillus niger
More LessA glycerol-kinase-deficient mutant of Aspergillus niger was isolated. Genetic analysis revealed that the mutation is located on linkage group VI. The phenotype of this mutant differed from that of a glycerol kinase mutant of Aspergillus nidulans in its ability to utilize dihydroxyacetone (DHA). The weak growth on glycerol of the A. niger glycerol kinase mutant showed that glycerol phosphorylation is an important step in glycerol catabolism. The mutant could still grow normally on DHA because of the presence of a DHA kinase. This enzyme, probably in combination with an NAD+-dependent glycerol dehydrogenase, present only in the mutant, is responsible for the weak growth of the mutant on glycerol. Enzymic analysis of both the mutant and the parental strain showed that at least three different glycerol dehydrogenases were formed under different physiological conditions: the NAD+-dependent enzyme described above, a constitutive NADP+-dependent enzyme and a d-glyceraldehyde-specific enzyme induced on d-galacturonate. The glycerol kinase mutant showed impaired growth on d-galacturonate.
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Stimulation of exoprotein secretion by choline and Tween 80 in Trichoderma reesei QM 9414 correlates with increased activities of dolichol phosphate mannose synthase
More LessAddition of choline (20 mm) or Tween 80 (0·06%) to the culture medium of Trichoderma reesei QM 9414 increased (a) the secretion of protein under both carbon-catabolite-repressed and -derepressed conditions, and (b) cellulase secretion under carbon-catabolite-derepressed conditions. In contrast, no stimulation by choline or Tween 80 was observed with the hypersecretory strain T. reesei RUT C-30. In view of the obligatory role of O-glycosylation in protein secretion by this fungus, an investigation was made into the effects on this process of choline and Tween 80. A membrane preparation was isolated from both strains of T. reesei and used to assay enzymes involved in O-glycosylation. Significant differences were observed with respect to the activity of dolichol phosphate mannose (Dol-P-Man) synthase only. Strain QM 9414, grown on media supplemented with choline or Tween 80 exhibited a two- to threefold higher activity of Dol-P-Man synthase compared to a control lacking these supplements. This stimulatory effect was observed during growth under both carbon-catabolite-repressed and -derepressed conditions. In contrast, strain RUT C-30 exhibited decreased activities of Dol-P-Man synthase when grown in media supplemented with choline. Choline had no effect on Dol-P-Man synthase in vitro, whereas Tween 80 decreased the activity. Thus the effect of Tween 80 or choline on protein secretion by T. reesei may be due to a stimulation of formation and/or activity of Dol-P-Man synthase, thereby elevating the level of O-glycosylation and protein secretion.
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Lactoferrin and transferrin damage of the Gram-negative outer membrane is modulated by Ca2+ and Mg2+
More LessLactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.
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Protein kinases in the entomopathogenic fungus Metarhizium anisopliae
More LessCyclic AMP (cAMP)- and Ca2+-dependent protein kinase activities of the fungus Metarhizium anisopliae were sought in extracts of ungerminated conidia, germinating conidia and mycelium, as well as in purified plasma membranes from mycelium. Ungerminated conidia contained a Ca2+/calmodulin-dependent protein kinase capable of phosphorylating multiple endogenous proteins and involved in triggering germination. cAMP-dependent protein kinase activity was not detected in ungerminated conidia in spite of the presence of cAMP in these conidia and the pre-germination synthesis of two cAMP-binding proteins. Most phosphorylation events in crude mycelial extracts were Ca2+-dependent but H-series inhibitors of cAMP-dependent kinases selectively repressed phosphorylation of a 27 kDA protein. Plasma membranes from mycelium contained a Ca2+-independent but H-8-sensitive protein kinase with multiple endogenous substrates for phosphorylation. 8-Azido[32P]cAMP bound selectively to a 52 kDa membrane protein indicative of a single cAMP-binding protein. Plasma membranes contained a phosphatase which rapidly (< 1 min) and selectively dephosphorylated a polypeptide of 15·5 kDa, thus being suited to cause rapid and reversible changes in membrane function. Membranes also contained an adenylate cyclase apparently involved in transmembrane signalling reactions, since mechanical or chemical treatments which stress the fungus caused rapid increases in intracellular levels of cAMP. Reconstitution experiments with a homogenate from a crisp-1 mutant of Neurospora crassa suggested G-protein regulation of Metarhizium plasmalemma adenylate cyclase.
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Alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803 after a salt shock
More LessSalt-induced change in protein synthesis were investigated in the cyanobacterium Synechocystis sp. PCC 6803. Immediately after a salt shock of 684 mm-NaCl, total protein synthesis was almost completely blocked. Then, parallel to the accumulation of the osmoprotective compound glucosylglycerol, protein synthesis recovered gradually but remained diminished. The activation of glucosylglycerol synthesis was not inhibited by chloramphenicol at concentrations which totally inhibited protein synthesis. The qualitative protein composition of salt-shocked and control cells was similar. However, the rates of synthesis of single proteins were altered in cells shocked for 10 h and adapted to high salt conditions. Using two-dimensional gel electrophoresis, proteins were found which were synthesized at enhanced rates after adding salt.
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The regulation of poly-β-hydroxybutyrate metabolism in Azospirillum brasilense during balanced growth and starvation
More LessActivities of the enzymes which are involved in the poly-β-hydroxybutyrate (PHB) cycle, in both synthesis and degradation reactions, were assayed in crude extracts of Azospirillum brasilense cells containing different amounts of PHB. The enzymes of the PHB cycle, of both the synthesis and the degradation process, were more active in PHB-rich cells than in PHB-poor cells. During 96 h of starvation of cells suspended in phosphate buffer, enzymes of the PHB cycle were more active in PHB-rich cells. There was a peak of activity of hydroxybutyrate dehydrogenase (BOHB-DH), β-ketothiolase and thiophorase after 24 h of starvation, due to polymer degradation. During the following hours of starvation there was a decrease in the activity of these enzymes. After 24 h of starvation the activity of acetoacetyl-CoA reductase dropped to a minimum level, because the cells could not synthesize PHB under these conditions. The specific activities of BOHB-DH, β-ketothiolase and thiophorase were higher in A. brasilense cells which were grown under low oxygen tension and consequently accumulated high levels of PHB, than in cells grown under high oxygen tension, with a low PHB content. Similarities to the pathway of PHB biosynthesis and degradation and its control in Azotobacter beijerinckii are described.
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An effective technique for enrichment and isolation of Candida cloacae mutants defective in alkane catabolism
More LessTechniques are described which allow mutated populations of Candida cloacae to be enriched efficiently (up to 167-fold in one round of enrichment) for mutants deficient in the alkane degradation pathway (Alk−). Such mutants, as well as being of scientific importance in studies of the degradation pathway, are also of commercial interest because several of the degradative intermediates are of value to the chemical industry. The Alk− mutants were readily isolated by their inability to grow on agar plates supplied with hexadecane as sole carbon source. A total of 288 Alk− mutants were isolated from, effectively, 4 x 106 mutagen-treated cells. They were further characterized by replica-plating using palmitic acid (PA) or acetate (Ac) as sole carbon source. Preliminary screening studies showed that of the 84 Alk− PA− Ac+ mutants, most could accumulate dicarboxylic acids from hexadecane and palmitic acid and at least one mutant also produced 3-hydroxyhexadecanedioic acid. Of the 80 mutants characterized as Alk− PA+, 16 produced small amounts of hexadecanol.
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Differences in the antigenic expression of immunomodulatory mannoprotein constituents on yeast and mycelial forms of Candida albicans
More LessThe expression of a strongly immunomodulatory mannoprotein complex (GMP) in the different forms of growth of the human commensal and opportunistic pathogen Candida albicans was studied using a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope of GMP. Immunofluorescence revealed that the surface of the yeast cells was highly reactive with mAb AF1, but that the reactivity was greatly reduced or disappeared during mycelial conversion. This modulation was shared by a number of strains of C. albicans, and was not solely a temperature- or nutrition-dependent phenomenon. Hypha-deficient strains (A12 and CA2) did not show variations of surface fluorescence under environmental conditions which were permissive for hyphal conversion (incubation in N-acetylglucosamine or Lee’s medium, at 37 °C). GMP extracts from yeast and mycelial forms of the fungus were separated into three chromatographically distinct, high molecular mass mannoprotein fractions (F1, F2 and F3), which were tested individually by indirect ELISA for mAb AF1 recognition. All yeast-derived constituents and two (F2 and F3) of the hyphal mannoproteins were recognized by the mAb. The low or absent reactivity of the F1 constituent from hyphal cells was confirmed by immunoblots. Irrespective of their source (yeast or mycelial), all fractions reacted to a similar extent with a polyclonal anti-Candida serum. Overall, the data suggest changes in epitope specificity and/or confinement of reactive constituents in the inner wall layers as possible mechanisms of modulated expression of mAb AF1-reactive epitope during mycelial conversion.
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Characterization and cloning of the gerC locus of Bacillus subtilis 168
More LessA Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to l-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 °C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector 𝜙105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6·3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2·9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentaly controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.
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Cloning and characterization of the gene for the “19 kD antigen of Mycobacterium bovis
Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in λgt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19–20 kDa. Gene expression occurred from the lac promoter in λgt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.
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Structure of a Rhodococcus gene encoding pigment production in Escherichia coli
More LessA 2·1 kbp DNA fragment from Rhodococcus strain ATCC 21145 gave rise to the production of blue and pink pigments in Escherichia coli when cloned downstream of a strong promoter. The sequence of this DNA fragment contains a single open reading frame with a putative ribosome-binding site, potentially coding for a single protein of M r 42 560. Deletion analysis and in vitro transcription-translation experiments support the hypothesis that pigment production in E. coli is due to a single enzyme whose catalytic activity is still unknown. This small pigment gene may become useful for the development of a new generation of chromogenic cloning vectors which do not require expensive substrates for the detection of gene expression.
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The occurrence and function of a-type cytochromes in the aerobic respiratory chain of Comamonas percolans NCTC 1937 grown under O2-sufficient and O2-limited conditions
More LessIntact cells of Comamonas percolans (NCTC 1937) after growth under O2-limited conditions in batch culture exhibit, in addition to b- and c-type cytochromes, an unusual pigment absorbing variably at 588 to 594 nm in reduced minus oxidized difference spectra at room temperature. CO difference spectra suggest ligand binding by this pigment and also reveal a prominent 448 nm absorption minimum and CO-binding b- and/or c-type cytochromes. Although the 588 to 594 nm-absorbing component is reminiscent of ‘cytochrome a 1’, claimed to be a terminal oxidase in some bacteria, O2-limited cells of C. percolans contain no detectable haem A. In contrast, cells grown under O2-sufficient conditions exhibit a membrane-bound cytochrome aa 3 and contain haem A. Low-temperature photodissociation studies of O2-sufficient cells show cytochrome aa 3 to be functional in CO and O2 binding and suggest that aa 3 is the terminal oxidase of a cytochrome b- and c-containing aerobic respiratory chain. Analogous studies of O2-limited cells reveal a component absorbing, in its unliganded state, at 448 nm. Exposure of such CO-liganded cells to white actinic light is followed by oxidation of b- and/or c-type cytochromes and, although the functional oxidase has not been identified, we conclude that C. percolans does not utilize a cytochrome oxidase of the a 1 type.
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Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis
More LessA tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1·1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1·1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10−7 per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10−8 per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9·5 and 11·0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.
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Further biological and molecular characterization of actinophage VWB
The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0·6 × 10−8 ml min1. The latent and rise periods were 140 and 100 min, respectively, and the burst size was estimated to be 130–250 p.f.u. Although phage VWB could infect only Streptomyces venezuelae ETH 14630 (ATCC 40755), of six different S. venezuelae strains tested, phage DNA could be introduced by transfection into most non-infectible strains. Upon transfection, phage DNA was propagated in these non-infectible strains and phage particles were released. In addition, the transfected strains could be lysogenized. By comparison of restriction fragments of VWB DNA, either free or integrated in the chromosomal DNA of the S. venezuelae ETH 14630 lysogen, the attachment site was localized. PAGE of the phage proteins revealed at least 17 different proteins with three major bands estimated as 16·5, 27·2 and 43 kDa in size. The N-terminal amino acid sequence of these supposed major head and tail proteins was determined. The corresponding DNA sequences on the phage genome were localized using oligonucleotides synthesized on the basis of the N-terminal amino acid sequences. The genes coding for the major structural proteins were shown to be clustered, as has been observed for other bacteriophages.
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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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