- Volume 139, Issue 6, 1993
Volume 139, Issue 6, 1993
- Microbiology Perspective
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- Biochemistry
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The enzymology of dicarboxylic acid formation by Corynebacterium sp. strain 7E1C grown on n-alkanes
More LessCultures of the Gram-positive bacterium Corynebacterium sp. strain 7E1C contained up to 300 mg dodecanedioic acid l−1 after growth on dodecane. Small amounts of tetradecanedioic acid (17 to 45 mg l−1) were produced during growth on tetradecane or methyl tetradecanoate. No dicarboxylic acids were detected after growth on hexadecane, hexadecanoic acid or 16-hydroxyhexadecanoic acid. Studies on the rates of degradation of exogenous dicarboxylic acids showed that this is not a significant factor influencing the accumulation of dodecanedioic and tetradecanedioic acids. The activities and substrate specificities of a number of enzyme activities involved in dicarboxylic acid metabolism were investigated. The specificities of the long-chain acyl-CoA synthetase and thioesterase, alcohol dehydrogenases and β-oxidation are consistent with the accumulation of dodecanedioic acid from dodecane and the lack of production of hexadecanedioic acid from hexadecane. The ω-hydroxy fatty acid may occupy a pivotal position in determining whether significant production of dicarboxylic acid occurs with this organism.
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Relationships amongst some bacterial and yeast lactate and mandelate dehydrogenases
Five yeast strains were isolated by enrichment culture on the basis of their ability to grow on mandelate and two of these strains were identified as Rhodotorula glutinis. In addition, a range of yeasts from culture collections was screened for growth on mandelate. The results suggest that mandelate utilization is a widespread but not universal characteristic within the genus Rhodotorula. Several of the yeasts contained an inducible NAD-dependent d(−)-mandelate dehydrogenase and an inducible dye-linked (presumably flavoprotein) l(+)-mandelate dehydrogenase. All the d(−)-mandelate dehydrogenases from the yeasts showed immunological cross-reactivity with each other (as judged by both immunoinhibition and immunoblotting), as did all the yeast l(+)-mandelate dehydrogenases that were tested. Determination of N-terminal amino acid sequences of several bacterial and yeast lactate and mandelate dehydrogenases, together with the evidence from the immunological studies, confirmed and extended previous proposals that there are several major groups of such dehydrogenases: FMN-dependent, membrane-bound l(+)-lactate and l(+)-mandelate dehydrogenases (M r = approx. 44000) in bacteria, mitochondrial flavocytochrome b 2 l(+)-lactate and l(+)-mandelate dehydrogenases (M r = approx. 59000) in yeasts, FAD-dependent, membrane-bound d(−)-lactate and d(−)-mandelate dehydrogenases in bacteria, and soluble NAD-dependent d(−)-mandelate dehydrogenases in both bacteria and yeasts.
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Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5
More LessA methyltransferase that acts on carminomycin and 13-dihydrocarminomycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycin-and baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5% yield with a purification of 114-fold in specific activity over the sample precipitated with 30–50% ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M r of about 41000. The enzyme was shown by gel filtration chromatography to have an M r of approximately 166000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-l-methionine typical of antibiotic methyltransferases; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-l-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, ε-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.
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Characterization of phosphofructokinase II and regulation of fructose 2,6-bisphosphate levels in Trichoderma reesei
More LessPhosphofructokinase II (PFK II) from Trichoderma reesei was partially purified (247-fold). The calculated K m values for fructose 6-phosphate and ATP were 0·7 mm and 40 μm, respectively. Upon incubation in the presence of [γ-32P]ATP, the enzyme formed a radioactive phosphoprotein with molecular mass of 67 kDa in autoradiography analysis after SDS-PAGE. Upon incubation in the presence of ATP-Mg and the catalytic subunit of cAMP-dependent protein kinase, its activity was not modified. The same result was obtained when a cell-free extract of T. veesei was incubated with ATP-Mg and cAMP. 2,4-Dinitrophenol caused a transient rise in cAMP levels in the fungal cell. These results provide evidence that the fructose 2,6-bisphosphate level in T. reesei is independent of cAMP concentrations and not related to a cAMP-dependent mechanism, but to the availability of substrate fructose 6-phosphate.
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Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus
More LessA proteinase has been purified from the stipes of senescent sporophores of the mushroom Agaricus bisporus. The proteinase was inhibited by PMSF. It has a broad pH optimum, 6·5–11·5, and a narrow substrate specificity, requiring both a hydrophobic amino acid in the P1 position and a minimum peptide chain length. The apparent molecular mass of the proteinase was 27 kDa when determined by SDS-PAGE and 14·1 kDa when measured by gel filtration. The isoelectric point of the proteinase was 9·0. Polyclonal antibodies have been raised to the proteinase. The proteinase from A. bisporus has similar properties to, and 60% N-terminal sequence identity with, proteinase K from the fungus Tritirachium album.
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- Biotechnology
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Processing and secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis
More LessThe gene encoding an immunodominant secreted antigen, MPB70, of Mycobacterium bovis was cloned into the plasmid vector pBluescript II KS+ along with its native ribosome-binding site. In this construct translation of the protein in Escherichia coli was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site. Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant E. coli. The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of E. coli. MPB70 was secreted into the periplasm of recombinant E. coli, where the 22 kDa form of the protein was predominant. The culture filtrate contained only the 22 kDa form of the protein, which was soluble. The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.
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- Development And Structure
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Differential compartmentation of ornithine decarboxylase in cells of Mucor vouxii
More LessA study was made to obtain an explanation for the differential effect in Mucorales of the ornithine decarboxylase (ODC) inhibitors 1,4-diamino-2-butanone (DAB) and α-difluoromethylornithine (DFMO), only the first of which affects cell differentiation; and for the paradoxical result that while it inhibits this process, DAB has no further effect on cell growth. Results obtained with cells permeabilized by two different methods which respectively do or do not affect internal membranes, and with protoplasts lysed under iso-osmotic conditions, suggest that the plasmalemma from M. rouxii cells is permeable to DAB, but not to DFMO, and that ODC is present in more than one cell compartment. ODC from the distinct cell compartments was differentially accessible to its substrate, and to DAB or DFMO, providing a reasonable explanation for the above results.
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- Environmental Microbiology
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Distribution of anaerobic fungi in the digestive tract of cattle and their survival in faeces
More LessA most probable numbers procedure was used to enumerate populations of anaerobic fungi in the digesta and faeces of cattle. Anaerobic fungi were isolated from the rumen, omasum, abomasum, small intestine, caecum, large intestine and faeces. By determining the amount of digesta in each organ of the digestive tract, it was possible to estimate the total population of anaerobic fungi in cattle and make comparisons between populations in different organs. In addition to enumerating anaerobic fungi in freshly collected samples, they were quantified in digesta and faeces which had been dried at ambient temperature and stored in air for up to 9 months. These experiments showed that a higher proportion of the anaerobic fungi present in the hindgut and faeces were able to withstand desiccation than those present within the gastric and pre-gastric organs. Our results support the hypothesis that the life cycle of anaerobic fungi consists of three stages; the motile zoospore, the vegetative thallus and an aero-tolerant survival stage (cyst or resistant zoosporangium).
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- Genetics And Molecular Biology
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Genotypes and phylogenetic relationships of Salmonella typhimurium are defined by molecular fingerprinting of IS200 and 16S rrn loci
More LessSummary: Molecular fingerprints of chromosomal genotypes in Salmonella typhimurium were generated by analysis of variation at the 16S rrn gene loci and the sites of the insertion sequence IS200. Genetic and reference strains of S. typhimurium were compared with clinical phage type strains from cases of human salmonellosis. Three 16S rrn profiles, one of which was predominant, were found. The copy number of the Salmonella-specific insertion sequence IS200 varied from 6 to 12, and all insertions were chromosomal. Three of the insertion sites shared by all strains were serovar-specific for S. typhimurium. Thirteen distinct profiles of IS200 were detected, providing a high level of intraserovar strain discrimination. Profiles were generally more conserved among genetic and reference strains; representatives of clinical phage type strains, which are recent human isolates, showed much greater diversity of IS200 profiles. Irrespective of their origin, strains could be assigned to IS200 profile groups, phylogenetically related lines identified by combinations of conserved insertion sites. Hybridization profiles of this mobile element are markers of intermediate and short-term evolution in S. typhimurium. They provide a fingerprinting scheme for the purposes of genetics, and delineate a molecular typing scheme for the purposes of epidemiology.
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Analysis of Borrelia species associated with Lyme disease by rRNA gene restriction fragment length polymorphism
More LessWe investigated the usefulness of rRNA gene restriction fragment length polymorphism (RFLP) for grouping of the Borrelia isolates associated with Lyme disease or from ixodid ticks. Genomic DNA was digested with a restriction enzyme, blotted and hybridized with an rrl (23S rRNA) gene probe. The sizes of the restriction bands showed a good correlation with the genotypes recently proposed, and Borrelia isolates of diverse geographic origin formed four distinct DNA groups. Group I included all of the USA isolates and some European isolates; group II contained European isolates and Asian isolates; group III comprised European and Asian isolates; group IV included Japanese isolates and an eastern Russian isolate. Groups I, II and III corresponded to Borrelia burgdorferi, B. garinii and group VS461, respectively. The RFLPs of Japanese isolates were rather divergent and some of the isolates were quite distinct from the USA and European isolates. RFLP analysis of the rRNA genes and flanking regions, using rrl gene probes as we reported here, may be useful in the taxonomic study of Borrelia.
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Multiple chromosomal and phenotypic changes in spontaneous mutants of Candida albicans
More LessPrevious studies have revealed the occurrence of multiple chromosomal alterations among spontaneous colony form mutants and clinical isolates of Candida albicans. In this report we show that such karyotype alterations are also seen in spontaneous and induced non-germinative mutants of the fungus. To determine if phenotypic changes other than colony form and microscopic morphology accompanied the rearrangements of the electrophoretic karyotype, we studied the following characteristics of the non-germinative and some of the colony form mutants: formation of pseudohyphae, chlamydospore production, germ tube formation, colony morphology, auxotrophy, growth at various temperatures, and colony morphology and pigment formation on selected media (bismuth sulphite and Phloxine B). We established that phenotypic and karyotypic variability among spontaneous, non-germinative mutants was no different than such variability amongspontaneous colony form mutants. Thus, non-germination may represent another phenotypicconsequence of genomic instability in C. albicans. The variability indifferent phenotypic attributes that occurred amongst the mutants was not associated with any given karyotype. Moreover, neither the low nor the high phenotypic variabilities observed were explained by the relatively high number of alterations in a limited number of chromosomes.
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Identification of two laccase genes in the cultivated mushroom Agaricus bisporus
More LessA cDNA library was constructed in λgt11 using mRNA from 11-d-old mycelium of Agaricus bisporus. Three clones containing laccase sequence were identified using an affinity-purified anti-laccase antibody. From one of these clones, a 333 bp sequence was used to identify further cDNA clones (including one which is close to full length) and a genomic clone. The coding sequences found were of two similar but not identical versions with differences at 36 out of 520 residues of deduced amino acid sequence. The laccase genes each encode a sequence expressed as a 2·3 kb mRNA, specifying a 520 residue polypeptide including a 19 amino acid residue signal peptide that is absent from the N terminus of the mature (extracellular) protein. The coding sequence of lcc1 is interrupted by 14 short introns. The lcc1 and lcc2 genes are not allelic as they do not segregate in uninucleate spores derived from a four-spored basidium. Comparison of the deduced amino acid sequences with that of the other fungal laccases that have been cloned, and with the very similar ascorbate oxidases from higher plants shows that whilst some sequence is absolutely conserved at and around the amino acid residues involved in copper binding, the overall sequence similarities are low.
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Transcriptional regulation of an endoglucanase and a cellodextrinase gene in Ruminococcus flavefaciens FD-1
More LessA gene which encodes a 35 kDa protein with both carboxymethylcellulase and xylanase activity was cloned from Ruminococcus flavefaciens FD-1 and the nucleotide sequence determined. The FD-1 gene, celE, and the celA gene, which encodes a cellodextrinase, were used as probes to analyse transcription in R. flavefaciens grown under different conditions. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose, although the effect was not as pronounced as induction by cellulose and was greater for the celA gene than for the celE gene.
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Cloning, sequencing and biochemical characterization of xylose isomerase from Thermoanaerobacterium saccharolyticum strain B6A-RI
More LessThe xylose isomerase gene from Thermoanaerobacterium saccharolyticum strain B6A-RI was cloned by complementation using Escherichia coli xyl-5 mutant strain HB101. One positive clone was detected and the recombinant plasmid, pZXI6, was isolated. The clone contained the vector pUC18 and an insert fragment of 4·5 kb. The cloned xylose isomerase gene (xylA) was expressed constitutively in E. coli. The gene contained one open reading frame (ORF) of 1317 bp, which corresponds to 439 amino acid residues. The molecular mass of the gene product was calculated to be 50474 Da from the deduced amino acid sequence. A putative promoter region (Pribnow box), TATAATATATAAT, which repeated twice at the −10 region in E. coli, was found 25 bp upstream of the ribosomal binding site. The deduced amino acid sequence of T. saccharolyticum strain B6A-RI xylose isomerase exhibited very high homology to those from Thermoanaerobacterium thermosulfurigenes 4B (formerly Clostridium thermosulfurogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). Codon usage in xynA, xynB and xylA showed a clear propensity for AT-containing isocodons. The native molecular mass of the purified recombinant thermostable xylose isomerase was 200 kDa, and the enzyme was a tetramer comprised of identical subunits. The apparent temperature and pH optima for activity of the cloned xylose isomerase were 80 °C and 7·0 to 7·5, respectively.
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Genetic organization, sequence and biochemical characterization of recombinant β-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI
More LessEndoxylanase (xynA) and β-xylosidase (xynB) genes from Thermoanaerobacterium saccharolyticum were subcloned from a cosmid clone (pXDM1) to generate pXPH3. The nucleotide sequence of a PstI-HindIII fragment in pXPH3 that contained xynB revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein. Another open reading frame (ORF1) of unknown function was found 21 bp downstream from the first stop codon of xynB. xynB, ORF1 and xynA had the same direction of transcription. xynB from T. saccharolyticum strain B6A-RI exhibited 45 % amino acid similarity, with 18 % amino acid identity to xynA of T. saccharolyticum strain B6A-RI, and 61 % similarity and 37 % identity with the β-xylosidase gene from Caldocellum saccharolyticum. Recombinant β-xylosidase was purified from E. coli (pXPH3) cells. The enzyme was a monomer with a molecular mass of 55 kDa. The specific activity and pH and temperature optima for hydrolysis of p-nitrophenyl-β-d-xylopyranoside (pNPX) were 5·53 U mg−1, 5·5 and 70 °C, respectively. The -xylosidase was stable at 65 °C, but lost activity at 85 °C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.
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β-Lactamase of Lysobacter enzymogenes: cloning, characterization and expression of the gene and comparison of the enzyme to other lactamases
More LessThe gene for the periplasmic β-lactamase of Lysobacter enzymogenes was isolated as part of a 1017 bp EcoRI fragment and the nucleotide sequence of the gene was determined. It has a G + C content of 71·5% and encodes a 27 amino acid signal sequence and the mature β-lactamase of 276 amino acids which has a mass of 29146 Da. The enzyme appears to be unique to L. enzymogenes but its amino acid sequence shows a high degree of homology with the amino acid sequences of the lactamase from Citrobacter diversus and other Class A β-lactamases. The -lactamase gene of L. enzymogenes was expressed in Escherichia coli using pUC118 as the vector. The production of active β-lactamase was highest after the active growth phase of the expression host and reached levels which were about three times higher than those obtained with L. enzymogenes.
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Structural analysis of the giant linear plasmid SCP1 in various Streptomyces coelicolor strains
More LessThe methylenomycin biosynthetic gene cluster was previously shown to be present on the 350 kb giant linear plasmid SCP1. The structures of SCP1 in various Streptomyces coelicolor A3(2) strains were analysed by pulsed field gel electrophoresis (PFGE). S. coelicolor A3(2) strains 1147, M138 and M146, previously genetically characterized as SCP1+, contained the free form of SCP1. S. coelicolor 1984 and 2106, earlier characterized as SCP1′-cysB and a stable cysD donor respectively, were found to contain very large linear plasmids, 550 kb and 1700 kb in size. Strain JCM4979, another SCP1+ derivative of A3(2), had a series of SCP1-related linear plasmids of 390630 kb. The integrated states of SCP1 in S. coelicolor SCP1-NF strains, an NF-like strain and a stable pabA donor were confirmed by PFGE, which revealed a lack of the intact SCP1 termini. In addition, a 70 kb linear plasmid was found in Streptomyces violaceoruber SANK95570, another methylenomycin producer. However, the plasmid was shown by hybridization to a methylenomycin resistance gene not to carry the methylenomycin gene cluster of this strain.
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Incompatibility properties of the narrow-host-range lactococcal plasmid pCI305
More LessThe cryptic plasmid pCI305 from Lactococcus lactis subsp. lactis UC317 was screened for incompatibility with a range of cloning vectors and was found to be incompatible with the theta-replicating plasmids pAM401 and pIL253. In contrast, lactococcal vectors containing the replication functions of the rolling circle replicating plasmids pSH71 and pWV01 were compatible with pCI305. A number of native lactococcal plasmids were also screened for both incompatibility and DNA sequence homology to the pCI305 replicon. A plasmid with high sequence identity was identified which was compatible with pCI305.
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Isolation and partial characterization of a new linear DNA plasmid isolated from Bacillus polymyxa SCE2
More LessA plasmid, designated pFS1, has been identified for the first time in a strain of Bacillus polymyxa. The plasmid is 16 kb in length. It is sensitive to digestion by DNAase I, restriction endonucleases and exonuclease III, but resistant to RNAase A and λ-exonuclease. A restriction map of the plasmid has been constructed, demonstrating its linear nature. The linear DNA molecules of pFS1 are attached at their 5′ ends to a protein which can be cleaved by Pronase. No homology was detected between pFS1 and chromosomal DNAs from a number of different B. polymyxa strains. Also, hybridization between pFS1 and total DNA from B. polymyxa SCE2 plasmid-cured derivatives does not occur. The plasmid seems to be cryptic since B. polymyxa strains harbouring pFS1 and the cured derivatives have the same phenotype.
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Sensitivity of naturally occurring coliphages to type I and type II restriction and modification
More LessProtection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution. There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems. Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria. To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems. The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage. The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate. On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E. coli and its accessory elements.
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- Pathogenicity And Medical Microbiology
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Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression
More LessCampylobacter jejuni strain 81116 contains two flagellin genes, flaA and flaB. Wild-type (WT) bacteria express flaA only, but flaB can be expressed under certain conditions. We have determined the importance of flagella for colonization of the avian caecum, which appears to be the natural environment for these bacteria. Mutants in which flaA or flaB, or both had been inactivated, and motility variants, were investigated. Flagella are not a requisite for colonization, but mutants lacking both flagellin genes colonized less efficiently than WT. Inactivation of the flaB gene, which had no effect on bacterial motility, enhanced chicken caecal colonization 1000-fold compared to WT. A variant (SF-1) with flagella composed of flagellin A, but with poor motility, also colonized better than WT. Conversely, mutants with an inactivated flaA gene colonized 100- to 1000-fold less efficiently than WT, regardless of their motility conferred by truncated or full-length flagellin B flagella. These results suggest that the presence of flagellin A, rather than motility, is essential for optimal bacterial colonization of chicken caeca.
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Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation
Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay,(c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U I−1) and specific activity (1749 U mg−1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.
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- Physiology And Growth
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Induction of extracellular proteinase in Candida albicans
More LessPulse-chase experiments indicated that the extracellular proteinase (EPR) of Candida albicans originates as a 45 kDa precursor protein which is processed to a 43 kDa protein prior to secretion. Secretion was routinely stimulated in EPR induction medium which contains bovine serum albumin (BSA) and glucose. Although EPR was not induced without glucose as a carbon source, EPR secretion was induced without the addition of BSA or other nitrogen sources. Furthermore, it was shown that EPR production was not induced at pH > 6·0, irrespective of the presence of a nitrogen source. This suggests that medium pH may act directly upon EPR induction, and not as a secondary effect of the nitrogen supply from EPR-mediated protein digestion, which exhibited a pH optimum of around pH 3·5. When germ tube induced cells were transferred to EPR induction medium, EPR was not induced. Thus, EPR production and germ tube formation may not be induced by the same conditions. We speculate that EPR production and germ tube formation do not co-operate in the invasive process but play different and separate roles.
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Alterations in the cellular envelope of spontaneous IIIMan L-defective mutants of Streptococcus salivarius
More LessIn Streptococcus salivarius, the phosphoenolpyruvate:mannose phosphotransferase system (PTSMan) transports and concomitantly phosphorylates mannose, glucose, fructose and 2-deoxyglucose. PTSMan consists of a membrane Enzyme II and two forms of Enzyme III (IIIMan) having molecular masses of 38·9 kDa (IIIMan H) and 35·2 kDa (IIIMan L) respectively. We have previously reported the isolation of spontaneous mutants lacking IIIMan L, and showed that they exhibited abnormal growth when cultured in mixtures of sugars containing glucose. The mutants also synthesize several cytoplasmic glucose-repressible proteins during growth on glucose and some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. We have now investigated the properties and composition of the cellular envelope of three S. salivarius IIIMan L-defective mutants (strains A37, B31 and G29) after growth on glucose. The mutants have altered sensitivity to various toxic compounds that interfere with cell-envelope functions. The mutants also exhibited altered membrane-protein profiles when analysed by two-dimensional PAGE and modified total lipid and phosphorus contents and lipid/protein ratio. In one mutant (strain G29), the proportion of the phospholipids separated by TLC was different from the parental strain. Electron microscopy indicated that one mutant (strain A37) possessed more fimbriae than the parental strain. The results suggested that these IIIMan L-defective mutants were affected in a global regulatory gene controlling several cellular or physiological functions, many of these being related to the cellular envelope.
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Involvement of the accessory gene regulator (agr) in expression of type 5 capsular polysaccharide by Staphylococcus aureus
More LessThe effect of an agr mutation on expression of type 5 capsular polysaccharide (CP) by Staphylococcus aureus Newman was investigated in different complex and synthetic media. CP expression by the agr mutant was strongly reduced in certain media but slightly in others, indicating that CP synthesis is positively controlled by agr. CP expression occurred in the post-exponential growth phase in both wild-type and mutant strains, suggesting that other regulatory systems could act in conjunction with agr.
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Oxygen inhibition of nitrogenase activity in Klebsiella pneumoniae
More LessA purpose-built oxystat has been used to study reversible inhibition of nitrogenase by O2 in the facultative anaerobe Klebsiella pneumoniae. C2H2-reducing activity in samples from either an anaerobic glucose-limited or an O2-limited diazotrophic chemostat culture was completely inhibited by exposure to a dissolved O2 concentration (DOC) of 1·5 μm or above. Subsequently, under anaerobic conditions, C2H2-reducing activity returned in the absence of de novo protein synthesis. The amount of activity returning never reached 100% of the initial anaerobic activity before O2 treatment. The degree of reversibility was inversely proportional to the log of DOC during exposure and was decreased by increasing the time of exposure to O2 (about 60% reversibility occurred after a 20 min exposure to 6 μm-O2). The failure to obtain complete recovery of activity was apparently not due to inactivation of the very O2-sensitive pyruvate-flavodoxin oxidoreductase (nifJ product) which provides electrons for nitrogenase activity in vivo. Samples from the O2-limited culture behaved similarly to those limited by glucose. Thus, ‘training’ of the organism to use O2 during growth does not influence the tolerance of nitrogenase to O2. Since the behaviour towards O2 reported here for K. pneumoniae differs from that known to occur in Aɀotobacter, the mechanism of protection of nitrogenase from O2 damage may differ in these organisms.
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The role of lipoic acid in product formation by Enterococcus faecalis NCTC 775 and reconstitution in vivo and in vitro of the pyruvate dehydrogenase complex
More LessThe role of the pyruvate dehydrogenase complex (PDC) in the formation of different fermentation products by Enterococcus faecalis was studied. This organism was grown on a semi-defined medium under various conditions in the presence or absence of lipoic acid, an essential cofactor of the enzyme complex. When grown on a medium without added lipoic acid, a very low activity, both in vivo and in vitro, of the PDC was observed. When pyruvate served as the energy source, lipoic acid was found to be essential for growth under anaerobic conditions at low culture pH values. The presence of lipoic acid in the culture medium had a marked effect on the production of acetoin: in the presence of lipoic acid, acetoin was produced only when the intracellular pyruvate concentration was relatively high, whereas in the absence of lipoic acid, acetoin was a common product. Under potassium-limited conditions, lactate was the main product and culture pH significantly affected the bacterial dry weight. After instantaneous addition of lipoic acid to a glucose + pyruvate-limited chemostat culture, an immediate activation of the PDC took place as deduced from the change in fermentation pattern. Reconstitution of the PDC by the addition of lipoic acid was also possible in cell-free extracts, although pre-incubation with ATP and lipoic acid for 90 min was necessary for maximal activation. The effects of an active PDC on product formation and the physiological role of the complex under anaerobic growth conditions are discussed.
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Microbial transformation of nitroaromatic compounds under anaerobic conditions
More LessThe transformation of several mono- and dinitroaromatic compounds (tested at 50–200 μm) by methanogenic bacteria, sulphate-reducing bacteria and clostridia was studied. Some of the nitroaromatics tested were transformed chemically by 1·5 mm quantities of culture media reducing agents, like cysteine or sulphide. This abiotic reduction occurred at the o-nitro-groups preferentially. Nitrophenols, p-nitroaniline and p-nitrobenzoic acid were completely transformed biologically into the corresponding amino derivatives. The nitroaromatics were transformed by all of the bacterial strains tested. While growing cells of sulphate-reducing bacteria and Clostridium spp. carried out nitroreduction, methanogen cells lysed in the presence of nitroaromatics. Nevertheless these culture suspensions converted nitroaromatics to the corresponding amino derivatives. This was also confirmed by crude cell extracts of methanogenic bacteria. The rate of nitroreduction by sulphate-reducing bacteria depended on the electron donors supplied and the cell density, with molecular hydrogen being the most effective donor of reducing equivalents. The toxicity of p-nitrophenol to some of the organisms tested depended on the concentration of the nitroaromatic compound and the type of organism.
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Inhibition of phosphatidylcholine and chitin biosynthesis in Pyricularia oryzae, Botrytis fabae and Fusarium graminearum by edifenphos
More LessColony growth of the fungi Pyricularia oryzae, Botrytis fabae and Fusarium graminearum was reduced by 50 % (ED50) by edifenphos concentrations of 7, 25 and 190 μm respectively; the phosphatidylcholine (PC) content of biomass of P. oryzae, B. fabae, and F. graminearum harvested from fungicide-containing-cultures was reduced by 50% by 6, 95 and 350 μm-edifenphos respectively. By contrast, the activities of membrane-bound chitin synthase preparations isolated from the three fungi were approximately equally sensitive to edifenphos. A direct relationship was observed between PC contents of biomass grown in the presence of ediphenphos and in vivo rates of chitin synthesis (biomass incubated with [3H]GlcNAc in the absence of fungicide). Membrane-bound chitin synthase preparations from P. oryzae grown in medium containing 3 or 6 μm-edifenphos had, at the same fungicide concentration, a lower rate of in vivo chitin synthesis than preparations isolated from biomass grown in the absence of edifenphos. Membrane-bound chitin synthase preparations from P. oryzae grown in the presence and absence of 6 μm-edifenphos had the same K m values for the substrate (UDP-[14C]GlcNAc) but different V max values. The results suggest that chitin synthesis is inhibited directly by non-competitive inhibition of chitin synthase activity, and indirectly following inhibition of PC biosynthesis. P. oryzae is very sensitive to edifenphos because inhibition of PC biosynthesis occurs at very low fungicide concentrations, and therefore in this fungus inhibition of PC biosynthesis probably represents the primary mode of action of the fungicide.
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- Plant-Microbe Interactions
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Aspartate transport in Rhiɀobium meliloti
More LessAspartate transport in Rhizobium meliloti was found to be mediated by at least two transport systems. High rates of aspartate uptake, necessary for growth on aspartate as a carbon source, required the dicarboxylate transport (Dct) system, which also transports succinate, fumarate and malate. The apparent K m for aspartate transport by this system was about 10 mm, compared to 15 βm for succinate. This difference in affinity was also apparent in competitive inhibition studies, which showed that succinate effectively inhibits aspartate transport. Although aspartate was not a preferred substrate, it was a very efficient inducer of the Dct system. Both the Dct system and a second aspartate transport system were capable of supplying aspartate for use as a nitrogen source. The second system had a lower apparent K m for aspartate transport (1·5 mm), and was competitively inhibited by glutamate. This aspartate-glutamate system was regulated independently from the Dct system, since it functioned in mutants lacking the Dct system regulatory genes dctB and dctD, and its induction did not coactivate the Dct system. Uptake kinetics in cultures growing on aspartate as nitrogen source showed rapid substrate exchange between extracellular and internal aspartate. R. meliloti was shown to be able to selectively activate the two uptake systems, and also regulated its metabolism as required to utilize aspartate as either carbon or nitrogen source.
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- Systematics
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Salmonella reference collection B (SARB): strains of 37 serovars of subspecies I
A reference collection of 72 strains representing 37 serovars of Salmonella subspecies I has been established for use in research on genetic and phenotypic variation in natural populations. Included are isolates of the host-adapted serovars S. choleraesuis, S. dublin, S. gallinarum, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. pullorum, S. sendai, S. typhi and S. typhisuis, as well as strains of S. enteritidis, S. typhimurium, and other commonly recovered serovars with broad host ranges. The isolates were characterized by enzyme electrophoresis for allelic variation in 25 chromosomal genes and represent 71 distinctive multilocus genotypes (electrophoretic types or ETs). Genetic relationships among the ETs are indicated in an evolutionary tree constructed by the neighbour-joining method from a matrix of Nei’s standard genetic distance.
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The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences
More LessPartial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.
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Phylogenetic diversity of the genus Cytophaga revealed by 16S rRNA sequencing and menaquinone analysis
More LessTo clarify the intra- and intergeneric relationships of the genus Cytophaga, 16S rRNA sequences and respiratory isoprenoid quinones were determined for the type strains of the 21 validly published species and one isolate in the genus Cytophaga. The sequence analysis revealed extreme heterogeneity of this genus, which diverged into nine distinct lines of descent. Each lineage of Cytophaga was characterized by possessing either menaquinone-6 (MK-6) or MK-7. The MK-6-possessing species were located in the two lineages that were remote from MK-7 species. One of the MK-6 lineages was composed only of terrestrial species and the other only of marine species. Flavobacterium aquatile, the type species of the genus Flavobacterium, was located in the MK-6 terrestrial lineage. The terrestrial Cytophaga species with MK-6 should be transferred to the genus Flavobacterium. The marine facultative anaerobes with MK-7 were located in the bacteroides branch, and possessed signature sequences with features intermediate between the bacteroides and the flavobacteria subdivisions. Cytophaga hutchinsonii, the type species of the genus Cytophaga, had a close relationship only with Cytophaga aurantiaca. The genus Cytophaga should be restricted to these two cellulose-degrading species. The genus Cytophaga is so heterogeneous that it should be divided into several genera and higher taxa in accordance with the phylogenetic relationships.
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Morphological, biochemical and molecular characteristics of Metarhizium anisopliae and M. flavoviride
More LessTwenty-four isolates of Metarhizium anisopliae and M. flavoviride, screened for virulence to desert locust (Schistocerca gregaria), were characterized by conidial morphology, API ZYM, isoenzyme electrophoresis and RFLP analysis of HaeIII digests on agarose minigels. Conidial morphology showed considerable overlap in measurements between the two species. However the API ZYM method showed that all isolates of M. anisopliae differed from those of M. flavoviride by possessing α-fucosidase activity. Isoenzyme electrophoresis clearly distinguished the two species by catalase and acid phosphatase banding patterns. M. anisopliae had a much simpler RFLP banding pattern than M. flavoviride, which showed > 20 bands, but individual isolates of both species had unique patterns. The methods used could all contribute to a unique ‘fingerprint’ for isolates of both species including M. flavoviride IMI 330189, which has been under intensive study for biopesticide development against locusts and grasshoppers.
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