- Volume 134, Issue 6, 1988
Volume 134, Issue 6, 1988
- Article
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Structuring Strain Data for Storage and Retrieval of Information on Fungi and Yeasts in MINE, the Microbial Information Network Europe
A distributed Microbial Information Network Europe (MINE) is being constructed by a number of major microbial culture collections in countries of the European Community, with the support of the Biotechnology Action Programme (BAP) of the Commission of the European Community. The representatives of the collections participating in MINE have agreed to adopt a general format for the computer storage and retrieval of strain data. This uniform format will facilitate the electronic combination and exchange of data from different collections in order to produce integrated catalogues and the use of identical commands to search the different databases. It is recommended to other collections who may wish to contribute data to the MINE network or between themselves.
Three kinds of records can be linked to the leading species records: strain records, synonym records, and alternative morphonym records. A minimum data set of 30 fields (similar to the fields used for producing catalogues) is defined that facilitates the exchange of data between the national nodes and serves as a directory to strains available at other nodes. It is suggested that the full strain record comprise 99 fields, grouped in 12 blocks: internal administration - name - strain administration - status - environment and history - biological interactions - sexuality - properties (cytology, biomolecular data) - genotype and genetics - growth conditions - chemistry and enzymes - practical applications. Several fields are divided into subfields of different ranks. Delimiters are used either to separate a range of entries that have to be indexed or to divide an entry from the reference to its source or remarks that should not be indexed. The contents and structure of the fields proposed for filamentous fungi and yeasts are described and in some cases illustrated by examples. Uniformity of input is essential for indexed fields and desirable for non-indexed fields. Seven thesaurus files are envisaged to ensure consistency.
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- Biochemistry
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Adenylylsulphate Reductase in a Dissimilatory Sulphate-reducing Archaebacterium
More LessThe recently described extremely thermophilic sulphate-reducing archaebacterium ‘VC-16’ has been shown to contain adenylylsulphate reductase (EC 1.8.99.2). The enzyme was purified 26-fold by ammonium sulphate fractionation, DEAE-cellulose chromatography, hydrophobic chromatography and preparative isoelectric focusing. The enzyme preparation gave a single band on analysis by SDS-PAGE. The M r was 160000 and the enzyme contained 1 mol FAD, 8 mols non-haem iron and 6 mols labile sulphide per mol enzyme. Ferricyanide was used as electron acceptor. The optimal reaction temperature was 85 °C under the assay conditions used and the pH optimum of the enzyme reaction was 8·0. The K m values for AMP and ferricyanide were 1 mm and 0·4 mm, respectively. The p1 of the archaebacterial adenylylsulphate reductase was 4·8.
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Purification and Properties of NAD-dependent Glutamate Dehydrogenase from Phycomyces Spores
More LessThe NAD-dependent glutamate dehydrogenase from Phycomyces spores was purified more than 300-fold. Estimation of M r by gel filtration gave a value of 98000 whereas after SDS-PAGE one major band of M r 54000 was found, suggesting that the enzyme is a dimer. The enzyme was virtually dependent on the presence of AMP for activity and showed half-maximal activation at 9·5 and 43 μm-AMP in the direction of amination and deamination respectively. ADP was nearly as effective at 20-fold higher concentrations. Other nucleotide monophosphates were ineffective and nucleoside triphosphates were slightly inhibitory. Hyperbolic kinetics were found for all substrates yielding K m values of about 10 mm for ammonium, 1 mm for 2-oxoglutarate and 0·1 mm for NADH in the direction of amination, and 10 mm for glutamate and 0·7 mm for NAD in the direction of deamination.
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Analysis of Sucrose Catabolism in Klebsiella pneumoniae and in Scr+ Derivatives of Escherichia coli K12
More LessIn contrast to a previous report, strains of Klebsiella pneumoniae were found to take up and phosphorylate the disaccharide sucrose via the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS). In addition to the two soluble and general components enzymeI and HPr of the PTS, a sucrose-specific enzymeIIScr (gene scrA), together with the enzymeIII, coded for by the gene crr, were needed for the vectorial phosphorylation of sucrose to generate intracellular sucrose 6-phosphate. This sugar phosphate is hydrolysed by a hydrolase (invertase, gene scrB) to generate glucose 6-phosphate and free fructose. The latter is converted to fructose 6-phosphate by an ATP-dependent fructokinase (gene scrK), an enzyme which is part of the sucrose and not of the fructose catabolic pathway. Analysis of different mutants of K. pneumoniae strain 1033, and of Escherichia coli K12 derivatives carrying R′scr plasmids isolated from K. pneumoniae, showed that the genes scrA, B, and K, together with a gene scrR for a repressor, form a genetic unit located on the chromosome of K. pneumoniae. These genes and the corresponding sucrose metabolic pathway are very similar to a previously described scr system encoded on plasmid pUR400 and found in other enteric bacteria.
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Evidence for a Glycosidic Linkage between Chitin and Glucan in the Cell Wall of Candida albicans
More LessThe alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of β-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and β-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.
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Lipid-linked Intermediates and the Synthesis of Acetan in Acetobacter xylinum
More LessSeveral strains of Acetobacter xylinum were screened for in vivo cellulose and acetan production, and for in vitro synthesis of a prenyl-diphosphate-hexasaccharide, using UDP-Glc, UDP-GlcA and GDP-Man as sugar donors. The lipid-bound saccharide was synthesized only by acetan-producing strains. Previous work has shown that the in vitro-synthesized lipid-linked saccharides have the same structure as the acetan repeating unit. The present results strongly suggest a precursor-product relationship. The strains that produced acetan lost their ability to do so by ageing of the culture.
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- Ecology
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Effect of Nitrate on Methane Production and Fermentation by Slurries of Human Faecal Bacteria
More LessMost probable number counts showed that denitrifying species were the numerically predominant NO− 3 reducing bacteria in the faeces of five methanogenic individuals [about 1010 bacteria (g dry wt faeces)−1]. In faecal slurries, however, denitrification was a relatively minor route of NO− 3 dissimilation, since only about 3% of the NO− 3 was converted to gaseous products, with NO− 3 being mainly reduced to NO− 2 and NH+ 4. When KNO2 was added to the slurries, denitrification became quantitatively more significant with approximately 23% of the NO− 2 being lost as gaseous products. The addition of KNO3 (10 mm) to slurries containing either starch or casein significantly decreased H2 and CH4 production. The effect of NO− 3 on methanogenesis was twofold: firstly, H2 accumulation decreased due to diversion of electrons towards NO− 3/NO− 2 reduction, and as a result of H2 being used as an electron donor for NO− 3 reduction, resulting in the removal of the methanogenic substrate; secondly, there was direct inhibition of methane-producing bacteria by NO− 3 and NO− 2. In starch-containing slurries, acetate: butyrate molar ratios were increased when NO− 3 was added but this effect was not observed when casein replaced starch. These results show that the ability of NO− 3/NO− 2 to act as an electron sink can significantly influence the major products of the human colonic fermentation.
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- Genetics And Molecular Microbiology
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Partial Nucleotide Sequence of 16S Ribosomal RNA Isolated from Armadillo-grown Mycobacterium leprae
More LessRibosomal RNA (rRNA) was isolated from Mycobacterium leprae recovered from infected tissue of the Nine-banded Armadillo, and nucleotide sequences near the 3′ end of the 16S species were determined by primer extension in the presence of dideoxynucleotides. Previously published data for bacterial 16S rRNAs show a pattern of conserved and non-conserved sequences that fit a common secondary structure. Our data for M. leprae fit this general pattern.
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Amplification of a Section of Chromosomal DNA in Methicillin-resistant Staphylococcus aureus following Growth in High Concentrations of Methicillin
More LessGrowth of two independently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) in increasing concentrations of methicillin (step-selection) resulted in increased resistance in these strains. When chromosomal DNA from the step-selected variants was probed using DNA sequences previously demonstrated to be associated with methicillin resistance in MRSA strains, amplification of the homologous chromosomal sequence was identified. Growth of these step-selected strains in the absence of methicillin resulted in loss of the amplified sequence, while the original sequence remained. There are differences between the two strains in the stability of maintenance of amplified sections. Prolonged storage of the variants on a high concentration of methicillin resulted in loss of amplified sections without concomitant loss of methicillin resistance. Thus amplification may be only one of at least two molecular mechanisms available to S. aureus to increase methicillin resistance in response to step-selection. Probing of cells of the highly resistant sub-population of a heterogeneously resistant MRSA strain showed that duplication of this mec-associated DNA is not involved in the mechanism of heteroresistance.
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The Expression in Staphylococcus aureus of Cloned DNA Encoding Methicillin Resistance
More LessA 4 kb fragment of chromosomal DNA was cloned from a clinical strain of methicillin-resistant Staphylococcus aureus. It comprises part of a section of the chromosome that was lost when the strain was cured of resistance to methicillin and to other antimicrobial agents. The fragment mediates an increased level of methicillin resistance when inserted into a shuttle vector and transformed back into the sensitive strain generated when the original DNA was deleted.
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Transfer of Genes Coding for Apoproteins of Reaction Centre and Light-harvesting LH1 Complexes to Rhodobacter sphaeroides
More LessSeveral methods of introducing mobilizable cloning vectors by conjugation into the photosynthetic bacterium Rhodobacter sphaeroides have been examined. The efficiency of transfer was sufficiently high to enable a bank of Rb. sphaeroides genes in Escherichia coli to complement non-photosynthetic mutants of Rb. sphaeroides, thus providing a generally applicable method of isolating Rb. sphaeroides genes. With a mutant incapable of synthesizing reaction centres and light-harvesting LH1 complexes as a recipient, the transfer of puf genes encoding reaction centre and light-harvesting LH1 polypeptides was examined in some detail. The spectroscopic and electrophoretic properties of this mutant and the newly photosynthetic transconjugant strain were consistent with the efficient transfer and expression of puf genes.
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Transposon Tn5 Mutagenesis of Genes Encoding Reaction Centre and Light-harvesting LH1 Polypeptides of Rhodobacter sphaeroides
More LessThe puf operon of the photosynthetic bacterium Rhodobacter sphaeroides encodes reaction centre and B875 (LH1) antenna polypeptides of the photosynthetic apparatus. This region of the genome was used to establish the applicability of random transposon Tn5 mutagenesis in this bacterium. Four Tn5 insertions have been mapped and one of the mutants characterized using a variety of techniques in order to establish that the fluorescence properties and polypeptide composition were consistent with the absence of reaction centre polypeptides. Following the ‘rescue’ of the transposon along with flanking regions of the puf operon, re-introduction of this construction into wild-type Rb. sphaeroides yielded the original mutation. This demonstrates that following homologous recombination, localized mutagenesis can direct Tn5 into a predetermined region of the Rb. sphaeroides chromosome. Accordingly, puf genes borne on plasmid pSRC2 were mutagenized with Tn5 in Escherichia coli, and the sites of insertion mapped physically. pSRC2 derivatives containing Tn5 were transferred to wild-type Rb. sphaeroides. puf genes have been mapped by correlating the photosynthetic properties of resulting strains with the sites of Tn5 insertion into pSRC2.
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Cloning and Oxygen-regulated Expression of the Bacteriochlorophyll Biosynthesis Genes bch E, B, A and C of Rhodobacter sphaeroides
More LessFour mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was transferred into these mutants using the helper plasmid pRK2073. Three clones that produced photosynthetic transconjugants from one or more of the bch mutants were isolated and characterized. These clones were used as probes to estimate levels of specific transcripts in cells undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7% of the eventual level of pigment had been synthesized.
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Transformation of the Cyanobacterium Synechococcus PCC 6301 Using Cloned DNA
More LessStable ampicillin-resistant transformants of Synechococcus PCC 6301 were isolated using as donor fragments chromosomal DNA cloned into ColEl-derived vectors. Using dark-incubated recipient cells, transformation was achieved reproducibly at frequencies of approximately 10−5 per cell and 3 × 102 per μg of donor DNA. These frequencies were 102- to 104-fold lower than those reported previously by other workers for the closely related strain PCC 7942 but only 5- to 10-fold lower than found by us in parallel experiments with both strains. Different donor fragments of PCC 6301 DNA gave different characteristic transformation frequencies which were greatly reduced by short internal deletions. The results are most simply explained by a model of integration of circular plasmid DNA into the recipient chromosome by a single crossover event.
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Integration of Replication-defective R68.45-like Plasmids into the Pseudomonas aeruginosa Chromosome
More LessR68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2·1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a ‘cut-and-paste’ mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely < 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.
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The Replication, Partition and yop Regulation of the pYV Plasmids Are Highly Conserved in Yersinia enterocolitica and Y. pseudotuberculosis
More LessThe replication genes (rep) of the virulence plasmid pYVe439-80 of Yersinia enterocolitica were localized and characterized by restriction endonuclease analysis. Comparison with pIBl, a virulence plasmid of Y. pseudotuberculosis, indicates that while the plasmids carry homologous rep genes their location with respect to the highly conserved ‘calcium region’ is different. This replication function is thermosensitive. Mini-derivatives of pYVe439-80 appear to be rather unstable. The region of pYVe439-80 containing homology to the incD determinant of F was shown to contain a plasmid-stabilization system (par). The region encoding par was characterized by restriction endonuclease analysis. pIBl contained an homologous par region but located differently. The pYV plasmids thus underwent rearrangements during their divergent evolution. While the positions of rep and par in the two plasmids are inverted with respect to the surrounding loci, our determination of the orientation of each locus rules out the hypothesis of a simple inversion of a quadrant of pYV. The gene encoding YOP5, a 26 kDa protein encoded by pIBl, was cloned on a mobilizable vector and introduced in Y. enterocolitica W22708 containing pYVe227 (indistinguishable from pYVe439-80), mutated in the homologous gene. The recombinant Y. enterocolitica secreted YOP5. Hence, the transcriptional activation and secretion systems of pYVe227 act on a yop gene from pIBl and on its product, indicating that these systems are interchangeable.
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Outer-membrane Permeability to β-Lactam Antibiotics in Yersinia enterocolitica
More LessTwo outer-membrane (OM) proteins of Yersinia enterocolitica YOMP-C and YOMP-F appear to function as porins. Mutants that were YOMP-C− and YOMP-F− exhibited changes in cephaloridine and [3H]glucose uptake and increased resistance to β-lactam antibiotics (especially cephalosporins) and tetracycline. Alterations in OM permeability may contribute to antibiotic resistance in Yersinia.
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Five Unique Temperate Phages from a Polylysogenic Strain of Bacillus thuringiensis Subsp. aizawai
More LessFive temperate phages were isolated from strain 4042B of Bacillus thuringiensis subsp. aizawai. The phages, which were heteroimmune, could also be distinguished by their host ranges, plaque and particle morphologies, serological specificities, and locations of restriction endonuclease cleavage sites on their chromosomes. Besides maintaining a stable lysogenic relationship with the 4042B host strain, each phage formed a stable lysogen with Bacillus cereus.
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A Complementation Analysis by Parasexual Recombination of Candida albicans Morphological Mutants
More LessBenomyl treatment (at 100 μg ml−1) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Y−phenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium.
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Molecular Cloning of a Gene Affecting the Autolysin Level and Flagellation in Bacillus subtilis
More LessA 2·8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HB101 and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRG1), of 10·2 kb, complemented flaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the HindIII and XbaI sites (1·0 kb apart) in pBRG1. The integration of a plasmid having chloramphenicol resistance closely linked to the flaD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the flaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.
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Molecular Cloning, Physical Mapping and Expression of the bet Genes Governing the Osmoregulatory Choline-Glycine Betaine Pathway of Escherichia coli
More LessAn analysis of the bet genes governing the osmoregulatory choline-glycine betaine pathway of Escherichia coli was performed. A 9 kb BamHI fragment, located 30 to 39 kb counterclockwise of the EcoRI site of lacZ, coded for all known Bet activities. The following genes were identified: the betA gene for the choline dehydrogenase, the betB gene for the betaine aldehyde dehydrogenase, and the betT gene or operon for the high-affinity choline transport. The betB and the betT genes were named in this paper, and the clockwise gene order was shown to be betA,B,T. Subcloning gave plasmids which expressed each of the three Bet activities separately. The cloned bet genes remained osmotically regulated, indicating the existence of several osmotically regulated promoters in the bet region. Salmonella typhimurium, which carried the bet region of E. coli in the broad-host-range vector pRK293 expressed the three Bet activities and displayed increased osmotic tolerance in the presence of choline.
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- Immunology
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Purification and Characterization of a 36 kDa Antigen of Mycobacterium leprae
More LessA 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47–9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.
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- Pathogenicity And Medical Microbiology
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Iron-regulated Outer-membrane Proteins of Escherichia coli Strains Associated with Enteric or Extraintestinal Diseases of Man and Animals
More LessThe SDS-PAGE patterns of the iron-regulated outer-membrane proteins from 70 strains of Escherichia coli isolated from various human and animal infections were analysed and the nature of the siderophores produced was examined. Iron-regulated 81 kDa and 74 kDa protein bands seen in SDS-PAGE gels were characterized further by immunoblotting using anti-81 kDa and anti-74 kDa (Cir) sera. The results showed considerable differences between the patterns of the iron-regulated outer-membrane proteins exhibited by the different strains. Nevertheless, three distinct and characteristic profiles, based on the most prominent bands expressed, could be identified, although not all strains produced patterns which matched with one of these. These results suggest the possibility of using the pattern of iron-regulated outer-membrane proteins expressed, as well as siderophores produced, as a new set of markers to characterize groups of pathogenic E. coli.
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Association of Treponema hyodysenteriae with Porcine Intestinal Mucosa
More LessThe association of Treponema hyodysenteriae with porcine caecal and colonic mucosal surfaces was studied by electron microscopy after orogastric inoculation of pigs with pure cultures. Examination of caecal and colonic mucosa from infected and control animals revealed that large numbers of the spirochaete were associated only with intestinal mucosal surfaces of infected animals. Further examination of the intestinal mucosa from infected pigs showed that T. hyodysenteriae colonized two sites preferentially: the mucus-filled crypts of Lieberkühn and the mucus gel covering the epithelium. Furthermore, no evidence of either specific or nonspecific adhesion to the epithelium proper was found, suggesting that penetration of, or trapping in the mucus gel may be the predominant mechanism of mucosal association by T. hyodysenteriae. Moreover, T. hyodysenteriae was also observed to be highly motile in intestinal mucus, moving faster than any other organism present, and this ‘high speed’ motility appeared to facilitate penetration into the mucosa. The pattern of motility observed was also highly suggestive of chemotaxis, and this was subsequently confirmed using an in vitro assay to porcine mucus material. It is suggested, therefore, that motility and chemotaxis are important factors/mechanisms in the association and colonization of porcine intestinal mucosa by T. hyodysenteriae.
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The Effect of Hormones on Trichomonas vaginalis
More LessThe hormonal milieu can alter susceptibility to infection. The effect of hormones on Trichomonas vaginalis was studied utilizing axenically cultured clinical isolates. Oestrogens, in physiological concentrations, decreased the growth of the organisms and their attachment to mammalian cells in vitro, and acted as a chemorepellent. The specificity of these effects was verified by their being blocked with anti-oestrogens, by the dose- and time-dependency of the responses, and by the lack of effect with other hormones. These results suggest that oestrogens may decrease the virulence of T. vaginalis; however, interactions between oestrogens and mammalian cells may promote the development of infection. Thus complicated interactions between hormones, micro-organisms and mammalian cells must determine whether exposure to oestrogens predisposes to or prevents the development of infection.
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Mouse Cachexia Induced by Trehalose Dimycolate from Nocardia asteroides
More LessTrehalose dimycolate (TDM) isolated from Nocardia asteroides induced in mice a severely wasted condition known as cachexia. Intraperitoneal injection of mice with five 10 μg doses of TDM in mineral oil at intervals of 2 d killed 90% of the animals within 26 d. Death followed a precipitous weight loss and an inflammatory process in the peritoneal cavity. When mice were injected intraperitoneally with a single 10 μg dose of TDM, 48 h later, they had begun to lose weight and exhibited extreme hypertriglyceridaemia and hypoglycaemia. Tumour necrosis factor (or cachectin) was detected in the plasma from animals injected with TDM. This cytokine released by mononuclear phagocytes may be involved in the induction of cachexia by TDM.
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Immunobiological Properties of Lipopolysaccharides Isolated from Fusobacterium nucleatum and F. necrophorum
More LessLipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the ‘LPS-nonresponsive’ C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.
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- Physiology And Growth
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Isolation and Physiological Characterization of Autotrophic Sulphur Bacteria Oxidizing Dimethyl Disulphide as Sole Source of Energy
More LessThe isolation of a number of strains of bacteria able to grow on dimethyl disulphide and dimethyl sulphide as sole source of energy is described. The isolates came from diverse habitats, including soil, peat, marine mud and a freshwater pond. The isolates were morphologically and physiologically best described as thiobacilli, capable of growth as Calvin cycle autotrophs on inorganic sulphur compounds, methylated sulphides or thiocyanate. They could not grow heterotrophically or methylotrophically. One isolate (E6) was examined in detail. Substrate oxidation kinetics indicated that methanethiol, sulphide, formaldehyde and formate, but not dimethyl sulphide, could be implicated as intermediates in dimethyl disulphide metabolism. Apparent K s values for the oxidation of dimethyl disulphide and methanethiol were 2.5 and 3·2 μm respectively. Growth yields in chemostat culture on dimethyl disulphide with and without thiosulphate indicated that energy conservation was probably coupled to the oxidation of formaldehyde and sulphide (derived from dimethyl disulphide via methanethiol) to CO2 and sulphate. Maximum growth yield (Y max) on dimethyl disulphide was 17 g cell-carbon per mol of dimethyl disulphide. At one dilution rate (0·078 h−1), the biomass of a culture limited by dimethyl disulphide increased when thiosulphate was also supplied, indicating a thiosulphate-dependent yield of 2·45 g cell-carbon mol−1. This is the first demonstration of the isolation of organisms into pure culture that are capable of growth on dimethyl disulphide as sole energy substrate, and of degrading it completely to CO2 and sulphate.
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Attraction of Agrobacterium tumefaciens C58C1 towards Sugars Involves a Highly Sensitive Chemotaxis System
More LessMotility of Agrobacterium tumefaciens C58C1 consisted of long straight runs, with relatively few tumbles. Speeds of up to 60 μm s−1 and runs of up to 500 μm were recorded. The propulsive mechanism appeared to resemble that of Rhizobium. Chemotaxis towards carbohydrates resolved four groups of sugars: chemoattractants with peaks at 10−6 m (sucrose, glucose and fructose); 10−5 m (maltose, lactulose and galactose); 10−4 m (raffinose, stachyose and arabinose); and weak or non-attractants (palatinose, lactose, cellobiose and xylose). In descending order, the magnitude of the responses was as follows: sucrose ⪢ maltose > lactulose > glucose > galactose/fructose > stachyose/arabinose/raffinose. The amino acids valine and arginine were good chemoattractants with peaks at 10−3 m, but no significant attraction was observed with alanine, cysteine, methionine or glycine. These results are indicative of a highly sensitive chemotaxis system towards sugars in A. tumefaciens C58C1, and suggest a role for this process in the ecology of the organism.
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C4-Dicarboxylate Metabolism in Free-living and Bacteroid Forms of Rhizobium leguminosarum MNF3841
More LessThe transport and catabolism of C4-dicarboxylic acids have been further studied in Rhizobium leguminosarum MNF3841. Uptake of [14C]succinate was induced in free-living cells of strain MNF3841 within 12 min of exposure to succinate and reached a maximum rate within 60 min. Free-living cells of strain MNF3841 oxidizing fumarate accumulated pyruvate when treated with arsenite, an inhibitor of pyruvate dehydrogenase. Generation of pyruvate from C4-dicarboxylates was accomplished by malic enzyme. Although malic enzyme was present in free-living cells grown on sucrose, higher activities were observed when fumarate or l-arabinose was the growth substrate. In crude extracts, malic enzyme activity required either NAD+ or NADP+ as cofactor, together with Mn2+, and was stimulated by K+.
Bacteroids from pea nodules transported [14C]succinate immediately after isolation, and contained twofold higher activities of both malic enzyme and pyruvate dehydrogenase than those found in fumarate-grown free-living cells. These data indicate that C4-dicarboxylates are available to nodule bacteroids and that they are catabolized through the tricarboxylic acid cycle using malic enzyme and pyruvate dehydrogenase to generate acetyl-CoA.
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Sterol Synergism in Paramecium tetraurelia
More LessParamecium tetraurelia is a naturally occurring sterol auxotroph with an absolute nutritional requirement for one of a small group of structurally related phytosterols. We report here a quantitative study demonstrating that a low, otherwise sub-supportive, concentration (≈0·020–0·050 g ml−1) of an essential phytosterol (stigmasterol) is adequate for growth of this ciliate, provided that a second, relatively non-specific sterol is available at a higher concentration (1·0 g ml−1) to allow for membrane biosynthesis. This phenomenon, referred to as sterol synergism, has been observed in a broad taxonomic range of organisms, with the conclusion that small amounts of specific sterols are required to perform some previously unknown, vital metabolic or regulatory function. Paramecium promises to be an excellent model organism for the elucidation of essential sterol function.
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A Haemoprotein Is Not Involved in the Control by Oxygen of Enteric Nitrogenase Synthesis
More LessStrains of Escherichia coli containing the Nif+ plasmid pRD1 were used to investigate the possibility that haem proteins are involved in the regulation by O2 of nif expression. Strains lacking 5-aminolaevulinate synthase (HemA−), and hence normally unable to synthesize haem proteins, showed an identical response to O2 in the presence or absence of added aminolaevulinate (and hence of haem proteins). It was concluded that the regulatory protein NifL is not a haem protein.
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Presence of Choline in Teichoic acid of Clostridium acetobutylicum N1-4 and Choline Inhibition of Autolytic Functions
More LessAddition of choline to growing Clostridium acetobutylicum led to abnormal cell septation, lack of cell separation and the consequent formation of chains. Similar results were obtained with the wild-type strain N1-4 and its autolysin-deficient mutant N1-4081. With strain N1-4, addition of choline at 1 to 2 mg ml−1 resulted in inhibition of autolysis assessed as autoplast formation in 0·6 m-sucrose, lysis by 0·3 m-NaCl/0·03 m-sodium citrate, lysis by 0·1% Triton X-100 and lysis by penicillin G. In vitro data confirmed the inhibition by choline of wall-degrading activity, using N1-4 cell walls as substrate. Choline was shown to be a component of the teichoic acid of C. acetobutylicum N1-4.
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Synthesis of Membrane and Periplasmic Proteins during Starvation of a Marine Vibrio sp
More LessChanges in membrane and periplasmic protein profiles induced by starvation conditions in the marine Vibrio sp. S14 were examined by one-dimensional gel electrophoresis. Analysis by densitometry resolved at least six periplasmic proteins, nine outer membrane proteins, and four cytoplasmic membrane proteins induced at various times during 120 h of nutrient and energy starvation. Eight of these were also synthesized by heat- and/or ethanol-shocked cells. Pulse-labelling indicated that the starvation-induced proteins were not products of degradation, and that their synthesis was differently modulated during starvation. The most pronounced changes occurred during the initial hours of nutrient and energy deprivation. The correlation between the initial changes in protein composition and utilization of the intracellular energy reserve poly-β-hydroxybutyrate is discussed. The rate of proteolysis during the initial hours of starvation was approximately 16 times greater than that during exponential growth.
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Effect of Growth Temperature on the Lipid Composition of two Strains of Thermus sp
More LessGrowth-temperature-dependent alterations in the total extractable lipid and polar lipid components of two strains of Thermus sp. isolated from a Portuguese hot spring were studied between 50°C and 78°C. The total extractable lipid varied between 8·0 and 10·6% of the cell dry weight; there were no alterations in the phosphorus and carotenoid content of the lipid extract, but the carbohydrate content increased as the growth temperature was raised. Three glycolipids and the four phospholipids were separated by thin-layer chromatography. The relative concentration of the major glycolipid (GL1) of strain SPS 17 (yellow-pigmented) increased with the growth temperature, whereas the relative concentration of a minor glycolipid (GL2) decreased. There were no temperature-dependent alterations in the relative concentrations of GL1 and GL2 from strain SPS 11 (colourless). The proportion of the major phospholipid (PL2) decreased in both strains as the growth temperature increased, whereas that of a minor phospholipid (PL1) increased. The major fatty acyl chains of both strains were 13-methyltetradecanoate (iso-C15) and 15-methylhexadecanoate (iso-C17). The GL1 in strains SPS 11 and SPS 17 had a glucose:glucosamine:glycerol:long fatty acyl chain ratio of 3:1:1:3.
† Present address: Departamento de Bioquimica, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain.
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Water Stress Plating Hypersensitivity of Yeasts: Protective Role of Trehalose in Saccharomyces cerevisiae
More LessWater stress plating hypersensitivity was studied in two strains of Saccharomyces cerevisiae, one of them being a mutant incapable of accumulating trehalose to significant levels. The wild-type strain was grown in a defined medium with glucose, maltose or ethanol as carbon/energy source. In each case plating hypersensitivity was demonstrated and resistance to the stress developed in the second half of the exponential growth phase. Development of resistance was accompanied by accumulation of trehalose and was apparently unrelated to glycerol content which, under these conditions, was always low. A qualitatively similar trend was observed in the mutant grown on glucose but trehalose levels remained low and recovery of stress resistance was only slight. Dinitrophenol induced trehalose breakdown in resting yeast and simultaneously induced the onset of plating hypersensitivity. A negative correlation was demonstrated between trehalose content and ‘plating discrepancy’ (log colony count on ‘normal’ agar – log colony count on stressing agar) for both strains under all experimental conditions. The correlation held for trehalose contents up to about 50 mg (g dry yeast)−1, above which the yeasts were apparently fully resistant. Trehalose was evidently a more effective compatible solute, per mole, than glycerol.
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The Rate and Topography of Cell Wall Synthesis during the Division Cycle of Escherichia coli Using N-Acetylglucosamine as a Peptidoglycan Label
More LessThe rates of synthesis of peptidoglycan and protein during the division cycle of Escherichia coli were measured by the membrane elution technique using cells differentially labelled with N-acetylglucosamine and leucine. During the first part of the division cycle the ratio of the rates of protein and peptidoglycan synthesis was constant. The rate of peptidoglycan synthesis, relative to the rate of protein synthesis, increased during the latter part of the division cycle. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Prior to the start of constriction the cell surface increases only by lateral wall extension. After cell constriction starts, the cell surface increases by both lateral wall and pole growth. The increase in surface area is partitioned between the lateral wall and the pole so that the volume of the cell increases exponentially. No variation in cell density occurs, because the increase in surface allows a continuous exponential increase in cell volume that accommodates the exponential increase in cell mass. The results are consistent with the constant density of the growing cell and the surface stress model for the regulation of cell surface synthesis. In addition, the elution pattern suggests that the membrane elution method does work by having the cells effectively bound to the membrane by their poles.
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- Systematics
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Outer Membrane Protein Pattern of Eubacterium plautii
More LessThe outer membrane SDS-PAGE pattern of Eubacterium plautii was characterized by a large number of surface exposed low- and high-molecular-mass proteins. Silver stainable carbohydrate was not present. The pattern was clearly distinct from those of outer membrane preparations of Eubacterium saburreum and Fusobacterium nucleatum. The results are compatible with a Gram-positive cell wall structure in E. plautii.
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Isolation and Identification of Thermotolerant Yeasts from Australian Sugar Cane Mills
More LessThermotolerant yeasts, many with the ability to ferment sucrose at temperatures above 40°C, were isolated from Queensland (Australia) cane sugar mills. Identification tests were done on 44 isolates and diagnostic keys were used to identify them. Of the thermotolerant isolates, 35 were identified as Kluyveromyces marxianus var. marxianus. Other thermotolerant strains were identified as Hansenula polymorpha, Geotrichum capitatum, Saccharomyces cerevisiae, Candida spp. and Debaromyces spp. Additional tests were done on these and on seven reference strains. Data for 75 characters for 51 strains were analysed using the simple matching coefficient and average linkage clustering. Six phenons were recovered that corresponded to the species named above.
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A Comparative Study of the Sugar Composition of Lipopolysaccharides Isolated from Vibrio cholerae, ‘Vibrio albensis’ and Vibrio metschnikovii
More LessA comparative study was made of the quantitative sugar composition of lipopolysaccharides (LPS) isolated from Vibrio cholerae (O1 and non O1 groups), ‘V. albensis’, ‘V. proteus’ and V. metschnikovii. The amino sugars 4-amino-4,6-dideoxy-d-mannose (perosamine) and 2-amino-2,6-dideoxy-d-glucose (quinovosamine) were present exclusively in LPS isolated from S-form O1 group of V. cholerae regardless of serotype (i.e. Ogawa or Inaba) and biotype (i.e. classical or eltor). Classical O1 group V. cholerae was distinguishable from eltor O1 group V. cholerae on the basis of the fructose content of the LPS: >3% and ≤1%, respectively. Distinct differences in the sugar composition of LPS were observed between V. cholerae and ‘V. albensi’, ‘V. proteus’ and V. metschnikovii.
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