- Volume 140, Issue 3, 1994
Volume 140, Issue 3, 1994
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Small cytoplasmic RNA of Bacillus brevis: transcriptional and phylogenetic analysis
More LessUsing a DNA fragment of Bacillus subtilis scRNA as a probe, a Bacillus brevis gene encoding the small cytoplasmic RNA was cloned and characterized. B. brevis scRNA consists of 273 nucleotides; the sequence has comparatively low homology (approximately 70%) with other Bacillus sequences. Phylogenetic analysis indicated that B. brevis forms a line of descent distinct from other Bacillus species. However, despite the low overall homology, both functional nucleotide sequence and secondary structural features defined among signal recognition particle (SRP) RNA family members were well conserved.
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- Review Article
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- Biochemistry
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Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: purification and characterization
More LessProline iminopeptidase (PepIP) is a major peptidase in Lactobacillus delbrueckii subsp. bulgaricus CNRZ397, encoded by the pepIP gene. Amplification and expression of this gene in Escherichia coli K12 resulted in a very high level of enzyme production. Moreover, export into the E. coli periplasm of 45% of PepIP activity allowed us to purify the enzyme easily by a single ion-exchange chromatography step. PepIP is a trimer of Mr 100000, composed of three identical subunits. In the presence of 0•1 % BSA, PepIP activity was optimal at pH 6–7 and stable at temperatures below 40°C. The enzyme was strongly inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, by bestatin and by heavy metal ions. It was also inactivated by p-chloromercuribenzoate, but was reactivated by adding dithiothreitol. PepIP is characterized by a high specificity towards di- or tripeptides with proline at the NH2-terminal position, but is not able to hydrolyse longer peptides, or peptides with hydroxyproline at the NH2-end. The NH2-terminal amino acid sequence of the purified PepIP corresponds to the amino acid sequence deduced from the nucleotide sequence of the pepIP gene.
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Ring cleavage reactions in the metabolism of (-)-menthol and (-)-menthone by a Corynebacterium sp.
More LessCorynebacterium sp. strain RWM1 grew with (-)-menthol, (-)-menthone and other acyclic monoterpenes as sole carbon sources. Growth on menthol was very slow, with a doubling time of more than 24 h, and was not rapid with (-)-menthone (doubling time 12 h). Concentrations of either carbon source greater than 0.025% inhibited growth. (-)-Menthone-grown cultures transiently accumulated 3,7-dimethyl-6-hydroxyoctanoate during growth, and (-)-menthol-grown cells oxidized (-)-menthol, (-)-menthone, 3,7-dimethyl-6-octanolide and 3,7-dimethyl-6-hydroxyoctanoate. Although neither a menthol oxidase nor a menthol dehydrogenase could be detected in extracts of (-)-menthol- or (-)-menthone-grown cells, an induced NADPH-linked monooxygenase with activity towards (-)-menthone was readily detected. With crude cell extracts, only 3,7-dimethyl-6-hydroxyoctanoate was detected as the reaction product. When the (-)-menthone monooxygenase was separated from an induced 3,7-dimethyl-6-octanolide hydrolase by chromatography on hydroxyapatite, the lactone 3,7-dimethyl-6-octanolide was shown to be the product of oxygenation.
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Purification and properties of a new exo-(1→3)-β-d-glucanase from Bacillus circulans YK9 capable of hydrolysing resistant curdlan with formation of only laminari-biose
More LessA (1.3)-β-d-glucan glucanohydrolase (EC 3.2.1.6), capable of hydrolysing resistant curdlan, was purified chromatographically from the culture supernatant of Bacillus circulans complex YK9 on Toyopearl HW-55F and butyl-Toyopearl 650M columns. The purified enzyme had a specific activity of 190 units mg−1 on regenerated curdlan. The molecular mass was estimated to be about 70 kDa as judged by SDS-PAGE. The enzyme had a pH optimum of approximately pH 6.0. It hydrolysed regenerated and resistant curdlans yielding predominantly laminari-biose, although the rate of hydrolysis of the former was much higher than the latter. This enzyme rapidly hydrolysed laminaran, curdlan and carboxymethyl-curdlan, but did not cleave schizophyllan and screloglucan, which have glucosyl side chains. The enzyme hydrolysed low molecular mass (1→3)-β-d-glucans (mean degree of polymerization,.DPTn = 131, 49 and 14) and laminari-heptaose more efficiently than curdlan. It also hydrolysed laminari-hexaose and -pentaose effectively, but laminari-tetraose only slightly and it did not hydrolyse laminari-triose or -biose. The enzyme is an exo-hydrolase of curdlan and various oligomers composed of (1→3)-β-d-glucosidic linkages, liberating laminari-biose from their non-reducing terminals. The laminari-biose generated was in the α-form.
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A newly isolated lectin from the Plant pathogenic fungus Sclerotium roltsii: purification, characterization and role in mycoparasitism
Jacob Inbar and Ilan ChetA novel lectin was isolated and purified from the culture filtrate of the soilborne plant pathogenic fungus Sclerotium rolfsii by anion-exchange chromatography using a DEAE-Sepharose column. The lectin came through column with the flow-through, whereas all the non-agglutinating proteins present in the crude preparation remained bound to the column until elution in a NaCI gradient. SDS-PAGE analysis of the agglutinating fraction revealed single band corresponding to a protein with a molecular mass of approximately 45 kDa. Agglutination of Escherichia coli cells by the purified lectin was not inhibited by any of the mono- or disaccharides tested, wheres the glycoproteins mucin and asialomucin did inhibit agglutination. Protease as well as 1,3-β-glucanase, were found to be totally destructive to agglutination activity, indicating that both protein and 1,3-β-glucan are necessary for agglutination. Using a biomimetic system based on binding of the lectin to the surface of inert nylon fibres revealed that the presence of purified agglutinin on the surface of the fibres specifically induced mycoparasitic behaviour in Trichoderma harzianum. Trichoderma formed tightly adhering coils, which were significantly more frequent with the purified agglutinin-treated fibres than with untreated ones or with those treated with non-agglutinating extracellular proteins from S. rolfsii. Other mycoparasite-related structures, such as appressorium-like bodies and hyph loops, were only observed in the interaction between T. harzianum and the purified agglutinin-treated fibres.
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- Biotechnology
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Manipulation of polyunsaturated, branchedchain and trans-fatty acid production in Shewanella putrefaciens strain ACAM 342
More LessShewanella putrefaciens strain ACAM 342 produced a range of polyunsaturated fatty acids (PUFA) including 18:2ω6, 18:3ω3, 18:3ω6, 18:4ω3 and 20:5ω3 (EPA). Under culture in Zobell's broth at 15 °C exponential-phase cultures produced 3.2 + 0.3% EPA, or 1.2 + 0.2 mg (g bacterial dry wt)-1. Alteration of growth phase, addition of biotin to the medium or a 10-fold decrease in nutrient levels had no significant effect (P>0.05) on levels of EPA. Growth in 7.0% NaCl medium markedly decreased the overall degree of fatty acid unsaturation (P<0.01), while growth on acetate as the sole carbon source increased the level of 18:2.6 from 0.4 + 0.1% to 7.1 % of total fatty acids through inhibition of branched-chain fatty acid synthesis. Addition of desaturase cofactors to the growth medium increased the proportion of EPA from 3.2 + 0.3% to 4.6%, but decreased the quantitative yield from 1.2. 0.2 to 0.9 mg (g bacterial dry wt)-1. This bacterium represents a model organism to study bacterial PUFA production. The trans-monounsaturated fatty acid 16:1ω7t was also produced under all culture conditions, together with four other trans-isomers, namely 14:1ω5t, 15:1ω6t, 17:1ωt and 18:1ω7t. Although the relative levels of trans-acids also changed under various culture conditions, there was no evidence that they were produced as a starvation or stress response.
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Dielectrophoretic characterization and separation of micro-organisms
More LessThe effective electrical conductivity values for a variety of Gram-negative and positive bacteria have been determined in the frequency range 10 kHz to 100 kHz. This information enabled experimental conditions to be selected where micro-organisms of different species can be separated using dielectrophoresis - the movement of particles induced by non-uniform AC electric fields. Mixtures of micro-organisms of different species were separated locally on a microscope slide using micro-electrodes of polynomial geometry, and their physical isolation into two separate suspensions was accomplished using a dielectrophoresis chamber that incorporated interdigitated, castellated micro-electrodes.
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- Development And Structure
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Tubular structures of Mycoplasma gallisepticum and their possible participation in cell motility
In five strains of Mycoplasma gallisepticum, a protein with a molecular mass of about 40 kDa was detected by immunoblotting with anti-pig brain tubulin polyclonal and monoclonal antibodies. In eight other mycoplasma species similarly tested no reaction was observed. Thin serial sections of M. gallisepticum and Acholeplasma laidlawii cells examined by transmission electron microscopy revealed a submembrane system of tubules in M. gallisepticum but not in A. laidlawii. The intracellular spatial distribution of the tubular structures was reconstructed. Thin sections of M. gallisepticum treated with anti-tubulin antibodies and colloidal gold particles (immunogold labelling) revealed distinct labelling of the tubular system. Analysis of the tubular structures by high resolution electron microscopy and optical diffraction showed their helical organization to be: diameter 40 nm, helix pitch approximately 20 nm and electron-transparent core 10 nm in diameter. A possible involvement of the tubular system in mycoplasma motility is suggested.
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- Environmental Microbiology
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Presence in rumen bacterial and protozoal populations of enzymes capable of degrading fungal cell walls
More LessRuminal bacteria and protozoa, and cell-free rumen fluid, were tested for the presence of enzymes involved in the degradation of the fungal cell wall. Protozoal homogenate obtained by ultrasonication showed chitinase (EC 3.2.1.14) and N-acetyl-β-glucosaminidase (EC 3.2.1.52) activities when assayed with fluorogenic 4-methylumbelliferyl substrates. The chitinase activity was predominantly of the ‘exo’-type. Lysozyme (EC 3.2.1.17) and 1,3-β-glucanase (EC 3.2.1.39) activities were also present in this fraction. All these activities, except lysozyme activity, were recovered mainly in the supernatant fraction of the homogenate (approximately 85% of the total activity). Lysozyme showed the same amount of activity in the precipitate and supernatant fractions. Bacterial homogenates had N-acetyl-β-glucosaminidase activity in both supernatant and precipitate fractions. The specific activity was one-third that of the protozoa. Bacteria able to grow in a medium with chitin as the sole carbon source were recognized and counted. Cell-free rumen fluid was unable to degrade any of the substrates tested.
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- Genetics And Molecular Biology
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Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds
More LessUsing translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK, and structural genes, nifH and fixA, in other fast-growing rhizobia whose nitrogen fixation regulation is less known. Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R. tropici, where clear symbiotic activation of either nifA or fixK expression could be observed. Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions. Only when the nifH promoter was in R. tropici and R. leguminosarum bv. phaseoli, was clear induction observed in the microaerobic free-living state. Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp, either in microaerobiosis or symbiosis. In contrast, the UAS region seemed to be unnecessary for fixA activation. However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp. The moderate induction observed in nitrogen-free medium only required the 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins. Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species.
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The gerB region of the Bacillus subtilis 168 chromosome encodes a homologue of the gerA spore germination operon
More LessSpores of gerB spore germination mutants of Bacillus subtilis 168 are defective in response to the germinative mixture of l-asparagine, glucose, fructose and potassium ions (AGFK), but are normal in the l-alanine (ALA) triggered germination response. A λ clone of 15 kbp carrying the gerB region has been identified. Sequencing of the gerB region of the clone revealed a cluster of three ORFs encoding putative proteins of 53.3, 41.3 and 42.4 kDa (GerBA, GerBB and GerBC, respectively). The first two of these proteins have substantial hydrophobic regions and the third is a possible lipoprotein. At least two, and probably all three products are required for normal germination in AGFK. The three proteins form a set of homologues of the products of the gerA operon, mutations in which cause a defect in the ALA germination pathway, but cause no defect in AGFK. The GerB proteins show 42%, 31% and 35% identity at the amino-acid level to the corresponding GerA proteins, and the homologues occur in the same order in both operons.
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Molecular characterization of pyrimidine biosynthesis genes from the thermophile Bacillus caldolyticus
More LessThe genes encoding the six pyrimidine biosynthesis enzymes from the thermophile Bacillus caldolyticus were characterized by cloning and complementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome, the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-pyrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr operon of Bacillus subtilis. Different parts of the B. caldolyticus cluster were cloned in two orientations in the expression shuttle vector pHPS9. Complementation studies in B. subtilis established that expression of the pyr genes was dependent on the vector-borne promoter, suggesting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase (pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosphoribosyltransferase (pyrE) levels in cells grown under different conditions indicated that expression of the operon is repressed 7–9-fold by addition of uracil to the growth medium. Based on the nucleotide sequence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.
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The lower pathway operon for benzoate catabolism in biphenyl-utilizing Pseudomonas sp. strain IC and the nucleotide sequence of the bphE gene for catechol 2,3-dioxygenase
More LessPseudomonas sp. strain IC is able to grow on biphenyl, 3-methylbiphenyl and 4-methylbiphenyl. These are converted to benzoate and the corresponding methylbenzoates. The lower pathway genes for the catabolism of the benzoates were cloned on a 22 kb HindIII fragment. Hybridization with gene-specific probes from the meta pathways of other catabolic plasmids showed that the gene order was identical to that of the operons carrying the same function from TOL plasmids. The nucleotide sequence of a 1241 bp region carrying the whole of the bphE gene (for a catechol 2,3-dioxygenase) and the 5′ end of the downstream bphG gene (for 2-hydroxy semialdehyde dehydrogenase) was determined. Both genes showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas strains. The upper pathway genes for the conversion of biphenyl to benzoate have also been cloned but no linkage with the lower pathway operon has been detected. Pseudomonas strain IC contains a large plasmid pWW110 (> 200 kb) and there are indications that this plasmid carries the bph genes.
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Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification
More LessThe gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2•1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2•1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.
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Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A
Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hydrophobic interaction and gel filtration chromatography, and reversed-phase HPLC. Three different active peaks (A1, A2 and A3) were detected, but the purified samples had identical N-terminal amino acid sequences for the first 15 amino acids as determined by Edman degradation analysis. Only a 2•4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino acids 5 to 13 of the amino acid sequence. The structural gene for carnobacteriocin A is located 600 base pairs into the 2•4 kb EcoRI fragment, but no other genetic information was detected on this unit. The structural gene includes an 18 amino acid N-terminal extension of the bacteriocin, ending with Gly-Gly residues in the −2, −1 positions with respect to the cleavage site. The bacteriocin consists of 53 amino acids that differ markedly from the majority of hydrophobic peptide bacteriocins characterized to date. Based on the amino acid sequence derived from the nucleotide sequence a molecular mass of 5052−85 Da was calculated. Mass spectrometry analysis showed that the molecular mass of the major component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 has a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.
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Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397
More LessThe proline iminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus is a major peptidase located in the cell envelope. Its structural gene (pepIP) has been cloned into pUC18 and expressed at a very high level in Escherichia coli to give a PepIP activity 15000-fold higher than that found in L. delbrueckii subsp. bulgaricus. The nucleotide sequence of the pepIP gene revealed an open reading frame of 295 codons encoding a protein with a predicted M r of 33006, which is consistent with the apparent size of the gene product. The amino acid sequence of PepIP shows significant homology with those of other hydrolases involved in the degradation of cyclic compounds. In particular, there is a region which includes an identified catalytic site containing a serine residue and a motif specific for the active sites of prolyloligopeptidases (Gly-X-Ser-X-Gly-Gly). The PepIP opens a new way for supplying cells with proline using the peptides resulting from the proteolytic degradation of caseins.
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Comparison of the electrophoretic karyotypes and chromosomal location of ten genes in the two varieties of Cryptococcus neoformans
More LessWe compared multiple isolates of the two varieties of Cryptococcus neoformans, as well as previously characterized representative isolates, for their electrophoretic karyotypes using pulsed-field electrophoresis. The two varieties could be clearly distinguished based upon the size of the smallest chromosome. The smallest chromosome for isolates of the gattii variety (serotypes B and C) was found to be 400–700 kb in size. The smallest chromosome for isolates of the neoformans variety was consistently found to be larger, approximately 770 kb in size. Isolates of the gattii variety averaged 13 chromosomes while the neformans variety averaged 12. The size of the Cryptococcus genome was found to be approximately 23 megabases. Isolates of C. neoformans var. neoformans tended to be more conserved than those of var. gattii with regard to gene position.
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Characterization of telomeric regions from Ustilago maydis
More LessThe isolation of telomeres from the phytopathogenic fungus Ustilago maydis is described. The telomeric repeat from the plant Arabidopsis thaliana, TTTAGGG, cross-hybridizes to Bal31-sensitive fragments of U. maydis DNA and detects many or all of the U. maydis chromosomes separated by pulsed-field gel electrophoresis (PFGE). This telomeric repeat was used to screen a library enriched for chromosome ends. Three clones were isolated which contained the tandemly repeated sequence TTAGGG. This sequence is identical to some known telomere repeats found in humans and other vertebrates as well as in some protozoa and moulds. In addition, the three telomeric clones had an almost identical 376 bp segment of middle-repeated telomere-associated sequences adjacent to the telomeric repeat. This segment hybridized to many or all U. maydis chromosomes separated by PFGE and showed a hybridization pattern in genomic digestions similar to that of the telomeric repeat. These results indicate that in U. maydis the same segment of telomere-associated sequences is located adjacent to the telomeric repeat in many or all chromosomes, which suggests that it may have a common role in chromosome function.
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Characterization of thermosensitive autolytic mutants from diploid Saccharomyces cerevisiae
More LessIn order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded and highly vacuolated cells (12%) and largesized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.
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- Physiology And Growth
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Stress tolerance and membrane lipid unsaturation in Saccharomyces cerevisiae grown aerobically or anaerobically
More LessSaccharomyces cerevisiae cells grown either aerobically or anaerobically were tested for tolerance to a brief heat stress (52°C, 5 min) or oxidative stress (20 mM H2O2, 15 min). Tolerance was related to growth phase, in that stationary phase cells were intrinsically more resistant to heat or oxidative stress than exponential phase cells. A mild heat shock (37 °C, 30 min) induced thermotolerance and oxidative tolerance in both aerobic and anaerobic cells. However, prior exposure to a low concentration of H2O2 (0.1 mM, 60 min) induced protection against the lethal concentration of H2O2 but not against the lethal temperature. Sensitivity to both heat and oxidative stress was dependent on membrane lipid composition. In the case of anaerobic cells, the most stress resistant had membranes enriched in saturated fatty acids, followed in order by cells enriched in oleic and linolenic acids. Aerobic cells with membranes enriched in palmitoleic and oleic acids showed the highest resistance to stress under all conditions. In both aerobic and anaerobic cells, a mild heat shock or oxidative shock induced markedly increased levels of thiobarbituric acid reactive substance (TBARS), indicative of malondialdehyde formation and lipid damage. Anaerobic cells with membranes enriched in linolenic acid had the highest TBARS, followed by cells enriched in oleic acid, with cells enriched in saturated fatty acids showing the lowest TBARS. The results suggest that heat and oxidative stress may share a common mechanism of damage through induction of oxygen-derived free radicals, resulting in membrane lipid damage. The extent of cellular damage was related to membrane lipid composition and correlated positively with increasing unsaturation of the phospholipid fatty acyl component.
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A nitrogen-limited, glucose-repressed, continuous culture of Saccharomyces cerevisiae
More LessGlucose-repressed growth of Saccharomyces cerevisiae was analysed in a nitrogen-limited continuous culture at different dilution rates (D). The glucose consumption of the yeast decreased from 3.4 g g-1 h-1 to 3.0 g g-1 h-1 when D was decreased from 0.3 h-1 to 0.15 h-1. No transcripts of the SUC2 and HXK1 genes, encoding, respectively, invertase and hexokinase isoenzyme 1, could be detected. Because both genes are regulated by glucose repression at the transcriptional level, this confirmed that the culture was glucose repressed at every D. During the decrease in D, no change in the activities or mRNA levels of key enzymes in carbon metabolism was observed, except for alcohol dehydrogenases I and II and phosphoglucomutase. These enzymes increased in activity and/or mRNA level when D was decreased, which was also observed in glucose- and galactose-limited continuous cultures. This demonstrates that the expression levels of alcohol dehydrogenases I and II, and also phosphoglucomutase, are coupled to the growth rate of the organism. A comparison between the alcohol dehydrogenase II activity in glucose- and nitrogen-limited continuous cultures demonstrated that the growth rate contributes as much to repression of alcohol dehydrogenase II activity as does glucose. Both the glucose consumption and the activity of the glycolytic enzymes were relatively constant when D was decreased and, as a consequence, the concentrations of intracellular metabolites remained constant. A slight decrease in the glucose 6-phosphate concentration was observed, which could be caused by the slight decrease in glucose consumption at low D values. The 2-oxoglutarate and cAMP concentrations increased twofold when D was decreased. The first probably reflects the increased NH4 consumption at high D values, while the latter is caused by the high amount of extracellular cAMP compared with the amount of intracellular cAMP. The decrease in growth rate raised the amount of biomass, while nitrogen was limiting in all cases. Analysis of the biomass composition at the different D values revealed that the amount of nitrogen per gram dry weight was constant, while the amounts of carbon, hydrogen and oxygen were higher at low D values. In addition, higher concentrations of trehalose and glycogen were found at low D values. This demonstrates that the glucose which is used at high D values for the production of biomass is converted into storage carbohydrates, e.g. trehalose and glycogen, at low D values. A nitrogen pulse did not result in a dramatic response of S. cerevisiae except for a rapid change in the free amino acid pool. Thus S. cerevisiae is not able rapidly to enhance growth upon a nitrogen pulse, which is in contrast with the response to a glucose pulse of a carbon-limited continuous culture of S. cerevisiae.
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Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae
Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh- strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh− strain [0.44 g biomass (g glucose)-1] was significantly lower than that of wild-type S. cerevisiae [0.52 g biomass (g glucose)-1]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of S. cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.
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Quantitative analysis of growth stimulation by glycine betaine in Salmonella typhimurium
More LessThe accumulation of compatible solutes, such as glycine betaine, is known to stimulate growth under conditions of osmotic stress. In Salmonella typhimurium the accumulation of glycine betaine is mediated by two osmotically activated transport systems, ProP and ProU. This study was undertaken to determine the quantitative relationship between glycine betaine accumulation from the environment and growth stimulation, and also the relative roles of the high affinity (ProU) and low affinity (ProP) transport systems. Our data show that relatively low concentrations of glycine betaine (= 10μM) are sufficient to stimulate growth and that under these conditions ProP and ProU transport systems are equivalent. At external concentrations of glycine betaine below 1 μM, cells able to express the ProU transport system possess a significant advantage over cells that only possess ProP. At high osmolarity the correlation between growth stimulation and cytoplasmic glycine betaine concentration is limited. At low glycine betaine concentrations further accumulation of the compatible solute stimulated growth. However, once the cells had accumulated 100 nmol glycine betaine per OD650 unit biomass no greater growth stimulation was observed in cells with higher levels of the compatible solute. The implications of these data for growth and pathogenicity of bacteria in natural ecosystems, such as foods, are discussed.
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Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination
More LessDifferent classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10λg ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.
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Interaction of the Ustilago maydis KP6 killer toxin with sensitive cells
More LessThe dsRNA viruses of the corn pathogen Ustilago maydis encode toxins that affect sensitive strains of the same species and related species. The KP6 toxin encoded by the P6 virus subtype is a binary toxin, consisting of two polypeptides, α (8-6 kDa) and β (9-1 kDa) with no covalent or hydrogen bonds between them. In this study the effect of each polypeptide was tested on sensitive and resistant cells and spheroplasts of U. maydis. The results indicate that both polypeptides bind to intact cells from sensitive and resistant strains; however, only spheroplasts of sensitive strains are affected by the toxin and both α and β are necessary to affect the spheroplasts.
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- Plant-Microbe Interactions
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Sequence and regulation of psrA, a gene on the Sym plasmid of Rhizobium leguminosarum biover phaseoli which inhibits transcription of the psi genes
More LessThe psr region of Rhizobium leguminosarum biovar phaseoli had originally been recognized on the basis of its ability to repress the transcription of the psi genes, one of which, psiA, inhibits exopolysaccharide synthesis when cloned in multi-copy plasmids. Both psr and psi are located on the symbiotic plasmid pRP2JI. The psrA gene was localized and sequenced. The deduced amino acid sequence of PsrA was shown to have similarity to the DNA-binding region of a family of other transcriptional regulators, consistent with its known effects on the expression of psi. The transcription of psrA itself appears to be constitutive in free-living Rhizobium, but is regulated by another gene on the Sym plasmid pRP2JI.
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Isolation and characterization of Rhizobium meliloti Tn5 mutants showing enhanced symbiotic effectiveness
A series of Tn5-induced mutants which showed enhanced symbiotic effectiveness (Eff++) was isolated from Rhizobium meliloti strains CXM1-105 and CXM1-188. Alfalfa plants inoculated by the Eff++ mutants had significantly higher shoot dry mass than plants inoculated by the parental strains. In the greenhouse, the most effective mutants increased the shoot dry mass of the host plants by 23-26% and plant total nitrogen by 23-27%. Interestingly, the frequency of the Eff++ mutants in strain CXM1-188 was higher than in the strain CXM1-105 (1.1% versus 0.4%); this was also the case for the auxotrophic mutants (1.2% in CXM1-188 versus 0.3% in CXM1-105). Genetic analysis of the mutants showed that the enhanced symbiotic effectiveness was cotransducible with Tn5. By the use of Southern hybridization and plasmid transfer, it was found that ten Tn5 insertions were located in the chromosome, five in megaplasmid 1, and six in megaplasmid 2.
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Virulence, growth, and surface characteristics of Erwinia amylovora mutants with altered pathogenicity
More LessErwinia amylovora CFBP1430 was mutagenized by phage MudllPR13 insertion Thirty prototrophic pathogenicity mutants with a single insertion were isolated. Among these, 17 non-pathogenic (Path-) mutants were obtained; 11 of them were unable to induce a hypersensitive response (HR) on tobacco (Hrp- mutants), whereas the remaining six were still able to do so (Dsp- mutants). Thirteen other mutants showed reduced virulence (Rvi-) and were still able to induce an HR. One of them appeared to be Path- on apple seedlings and Rvi- pear seedlings. All the Hrp- mutants and all but three of the Dsp- mutants mapped in the hrp-dsp gene cluster previously reported. Some Rvi- mutants also proved to map in this hrp-dsp region; most of them, as well as two Dsp- mutants map in an unknown genomic region. Cell-surface components thought to play a role in bacterial pathogenicity were examined, including exopolysaccharides (EPS), lipopolysaccharides (LPS), and outer-membrane proteins. One mutant only was found to be non-capsulated and unable to produce EPS. The insertion in this mutant mapped in a genomic cluster involved in amylovoran synthesis. Unlike the parental strain, some mutants exhibited sensitivity to the phage Ffm and this phenotype was associated with a modified LPS electrophoretic profile. Rvi- mutants and some Path- mutants were able to multiply in planta to some extent; other Path- mutants reached only a low population level, except the non-capsulated one, which rapidly decreased to an undetectable level.
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- Systematics
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Small cytoplasmic RNA of Bacillus brevis: transcriptional and phylogenetic analysis
More LessUsing a DNA fragment of Bacillus subtilis scRNA as a probe, a Bacillus brevis gene encoding the small cytoplasmic RNA was cloned and characterized. B. brevis scRNA consists of 273 nucleotides; the sequence has comparatively low homology (approximately 70%) with other Bacillus sequences. Phylogenetic analysis indicated that B. brevis forms a line of descent distinct from other Bacillus species. However, despite the low overall homology, both functional nucleotide sequence and secondary structural features defined among signal recognition particle (SRP) RNA family members were well conserved.
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