- Volume 139, Issue 7, 1993
Volume 139, Issue 7, 1993
- Biochemistry
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Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1
More LessSummary: Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases. APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.
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Luteolin absorption in Rhizobium meliloti wild-type and mutant strains
More LessSummary: Luteolin is a flavonoid produced by plants which is required for induction of nod genes in Rhizobium meliloti. R. meliloti absorbed luteolin at higher rate than all other bacteria tested, including R. leguminosarum. The flavonoids naringenin and quercetin, which do not induce the expression of nodulation genes of R. meliloti, were absorbed at a lower rate by this species, suggesting a certain degree of species specificity of flavonoid absorption. Luteolin accumulated preferentially in the outer membrane, but a small amount was always found in the inner membrane. Luteolin strongly inhibited NADH oxidase, an enzyme of the respiratory chain, raising the possibility that the site of luteolin absorption in the outer membrane allows the protection of the respiratory chain located in the inner membrane from an excess of flavonoids. The incorporation of luteolin was found to be affected in some exo or nod mutants of R. meliloti. The exoB mutant, which does not produce exopolysaccharides, accumulated lower amounts of luteolin in the outer membrane than the exo + parent. Among the nod mutants affected in nodulation genes, those not expressing any of the three nodD genes accumulated luteolin at a significantly lower level in both the outer and the inner membrane. A strain overexpressing the nod genes, particularly the nodD genes, absorbed luteolin at a higher level in both membranes. These results indicate that absorption of luteolin by R. meliloti involves several gene products, including the NodD protein.
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Phylogeny among the basidiomycetous yeasts inferred from small subunit ribosomal DNA sequence
More LessSummary: The sequence of the small subunit nuclear ribosomal DNA (18S rDNA) was determined for seven selected species of the teliospore-forming yeasts and Filobasidiaceae in the basidiomycetous yeasts. A phylogenetic tree, including published reference sequences, was inferred from 1623 sites which could be unambiguously aligned. The molecular phylogeny, using a chytridiomycete as an outgroup, divided the eight basidiomycetous yeasts into two groups which correlated well with both septal ultrastructure (simple pore or dolipore) and cellular xylose (present or absent). The first group included the teliospore-forming yeasts Rhodosporidium toruloides and Leucosporidium scottii, and Erythrobasidium hasegawianum, which is currently a member of the Filobasidiaceae. The second group was formed by the filobasidiaceous yeasts, comprising a Cystofilobasidium capitatum/Leucosporidium lari-marini/Mrakia frigida branch, and a Filobasidium floriforme/Filobasidiella neoformans branch. Our molecular data support the principal chemotaxonomic and ultrastructural evidence, which indicates a very close affinity between C. capitatum and L. lari-marini.
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Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism
More LessSummary: The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived 64Cu. Cells pre-incubated with 10 m-Cu2+ accumulated 64Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was non-exchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t1/2 % 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). Zn2+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper. This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak Pv) of the column used to size-fractionate copper-binding species. The metal in Pv was exchangeable in vivo and in vitro. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20 % of the 64Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily cell-dependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the non-exchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of 64Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphide-based mechanism of copper resistance in this yeast.
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Distribution of 64Cu in Saccharomyces cerevisiae: kinetic analyses of partitioning
More LessSummary: The cell association of copper in the yeast Saccharomyces cerevisiae can involve both binding to the cell wall and the accumulation of copper within the cell. The former process requires the concurrent generation of H2S by the cell via the reduction of sulphate. The contributions of each of these processes to the uptake of 64Cu by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected. The Michaelis constant for uptake (4 m) is independent of the type of cell association which is occurring, suggesting, although not requiring, that both processes are associated with a common kinetic intermediate. The time dependence of the cell-association of 64Cu also suggests the presence of this intermediate pool of bound copper. The Vmax for uptake includes a constant contribution from accumulation of 64Cu within the plasmalemma [0.1 nmol min-1 (mg protein)-1] plus that fraction of the 64Cu within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide. This latter contribution to V max can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the 64Cu2+ uptake buffer. Both processes are energy-dependent although the sulphide-dependent periplasmic accumulation is somewhat more sensitive to metabolic inhibition. This can be attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide. Periplasmic 64Cu accumulation is strongly inhibited by Zn2+ and Ni2+. This inhibition is due to competition for cell-generated sulphide; in the presence of 64Zn2+, the decrease in 64Cu bound is quantitatively related to the amount of 65Zn which becomes cell-associated. In contrast, intracellular 64Cu uptake is not inhibited by these two metals (at 50 M) showing that the copper translocation pathway is metal-specific. These observations suggest a model for the way newly arrived copper is handled at the cell membrane and is partitioned for intracellular uptake.
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Purification and partial characterization of β-glucosidase from plasmodial membrane and culture medium of Physavum polycephalum
More LessSummary: β-Glucosidase activity was detected in the plasma-membrane fraction of plasmodia of Physavum polycephalum. The enzyme activity was also found in the extracellular (medium) fraction after maximal growth had been attained, though the activity in this fraction was negligible during the exponential growth phase. The specific activities in both fractions were increased appreciably when the plasmodia were cultured in a glucose-free medium. The β-glucosidase was purified from the culture fluid by gel-filtration, ion-exchange and hydrophobic chromatographies. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis, and the molecular mass was estimated to be about 65 kDa. The enzyme showed highest activity at pH 4·5 and at 55 °C. The β-Glucosidase from the membrane fraction showed very similar properties to that from the culture medium, suggesting that the extracellular enzyme was derived from the membrane. The enzyme was active on laminarin and lichenan as well as on p-nitrophenyl-β-D-glucoside; it was strongly inhibited by D-glucono-l,5-lactone, N-bromosuccinimide and Hg2+.
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- Biotechnology
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Development of an electroporation procedure for gene disruption in Lactobacillus helveticus CNRZ 32
More LessSummary: An electroporation-mediated transformation method was developed and optimized for Lactobacillus helveticus CNRZ 32. The effects of electroporation buffers, growth conditions, cell-wall-weakening agents and field strength on transformation frequency were examined. Optimal conditions yielded a frequency of 2 104 transformants (g pGK12)-1. Using this procedure, an integration plasmid was introduced into L. helveticus CNRZ 32 to inactivate the chromosomal X-prolyl dipeptidyl aminopeptidase gene (pepXP). The integration plasmid, designated pSUW200, consisted of pUC19, the pE194 erythromycin resistance gene and a 1.6 kb internal fragment of the pepXP gene. The erythromycin resistance transformants obtained were X-prolyl dipeptidyl aminopeptidase (X-PDAP) negative. Southern hybridization results indicated that pSUW200 had integrated into the L. helveticus CNRZ 32 chromosome via Campbell-type integration. After growth of the pSUW200-derived transformants for 112 generations under non-selective conditions, erythromycin-sensitive X-PDAP+ isolates were obtained. Southern hybridization results indicated that precise excision of pSUW200 had occurred via recombination between the 1.6 kb pepXP-derived nontandem repeats.
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- Development And Structure
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Peptidoglycan synthesis in Salmonella typhimurium 2616
More LessSummary: HPLC analysis of peptidoglycan synthesis in Salmonella typhimurium strain 2616 has revealed that: (i) there is observable variation in the composition, but no significant variation in the overall degree of cross-linking, of newly synthesized peptidoglycan during the division cycle; (ii) the types of muropeptide that constitute peptidoglycan do not vary over a wide range of growth rates; and (iii) the composition and maturation kinetics of S. typhimurium peptidoglycan are, as expected, similar to those of Escherichia coli. We propose a unitary model of peptidoglycan synthesis, with single-strand incorporation occurring at both side wall and polar sites.
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- Environmental Microbiology
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Effects of acidophilic protozoa on populations of metal-mobilizing bacteria during the leaching of pyritic coal
More LessSummary: Five acidophilic protozoa (three flagellates, one ciliate and one amoeba) were isolated from acid mine water and a coal biotreatment plant, and grown in mixed cultures with acidophilic bacteria. Cultures were routinely maintained in ferrous sulphate media: in media containing pyrite or pyritic coal, protozoa grew in cultures containing coarse-grain (61-200 μm) but not fine-grain (< 61 μm) minerals. In cultures of pyritic coal, protozoa grazed iron-oxidizing and heterotrophic bacteria, but to varying extents. One of the flagellates appeared only transiently in coal leachates, whilst the four others persisted through the 100 d incubation. A reduction of numbers of unattached iron-oxidizing acidophiles, due to protozoan grazing, did not always result in lower rates of pyrite oxidation. The presence of protozoa was noted to effect changes in acidophilic populations, in particular often causing Leptospirillum ferrooxidans to become the dominant iron-oxidizer at an earlier stage than in corresponding protozoa-free controls.
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Effect of previous growth conditions on the starvation-survival of Escherichia coli in seawater
More LessSummary: The starvation-survival of Escherichia coli in seawater was assessed by plate and epifluorescence counts, 3H-label decrease, cellular DNA concentrations, and metabolic activities. These assays were performed on two types of populations, adapted and non-adapted to seawater. The number of viable cells in the adapted population remained constant throughout starvation-survival in sterile seawater. In contrast, a significant decrease in the ability of the non-adapted E. coli to form colonies on plates following starvation-survival in sterile seawater was observed. However, this drop in viable counts was not mirrored by the epifluorescence counts and 3H-label, which did not show major changes for either population during the experiments, indicating maintenance of the number of cells. In addition, a significant increase in and subsequent maintenance of DNA content and thymidine incorporation was observed for both populations during starvation-survival in sterile seawater. The changes in cell-attached exoproteolytic activity and electron transport system activity showed that adapted and non-adapted E. coli cells maintain their metabolic potential. Cell-free exoproteolytic activity was drastically reduced in both populations. Adapted cells showed higher electron transport system activity and thymidine incorporation than non-adapted cells at the onset of starvation-survival. The effect of previous adaptation on E. coli starvation-survival, as assessed by plate counts and 3H-label decrease, w as also observed in raw seawater. It seems from these data that the biological potential of E. coli cells suspended in sterile seawater has not been switched off or impaired seriously.
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- Genetics And Molecular Biology
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Sequence and mapping of the aroA gene of Staphylococcus aureus 8325-4
More LessSummary: The aroA gene of Staphylococcus aureus 8325-4 was cloned. Sequence analysis and the phenotype of directed plasmid insertions 5” to aroA suggest that aroA is located in an operon and that it maps 3” to the aroC and aroB genes. A revised consensus sequence for the aroA gene product EPSP synthase binding site for its substrate (phosphoenolpyruvate) and an inhibitor (glyphosate) is proposed. An aroA insertion mutant isolated by allelic replacement was employed in genetic mapping experiments which demonstrated the gene order thy aroA tyrB in SmaI fragment A of the S. aureus 8325-4 chromosome. The aroA::Tcr mutant required aromatic amino acids but remained independent of p-aminobenzoic acid (PAB). This could be due to the insertion being located close to the 5” end of the gene, allowing expression of a truncated protein. The PAB independence may explain the finding that the mutant was not attenuated in mouse infection experiments. It was not possible to isolate a null mutant in aroA.
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pRj5: a naturally occurring Staphylococcus aureus plasmid expressing constitutive macrolide—lincosamide—stireptogramin B resistance contains a tandem duplication in the leader region of the ermC gene
More LessSummary: The 2.55 kb Staphylococcus aureus plasmid, pRJ5, confers constitutive resistance to macrolide–lincosamide–streptogramin B (MLS) antibiotics. pRJ5 is nearly identical to the inducible MLS resistance plasmid pT48, and has homology with the S. aureus plasmids pE194 and pSN2. The HindIII-C and/or Hind-B fragments were required for stable maintenance of the plasmid and probably carry palA. Plasmids pRJ5 and pT48 were shown to belong to the same incompatibility group, Inc12 (L). DNA sequencing showed that pRJ5 contains a 28 bp direct tandem duplication in the leader/attenuator region of ermC. This is likely to change the secondary structure of the methylase mRNA, allowing constitutive expression of ermC. The type of mutation found on plasmid pRJ5 is different from those observed in similar 2.5 kb constitutive MLS-resistance plasmids isolated from other Gram-positive bacteria, including staphylococci.
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Cloning and nucleotide sequence of the Butyrivibrio fibrisolvens gene encoding a type III glutamine synthetase
More LessSummary: A Butyrivibrio fibrisolvens glnA gene encoding glutamine synthetase (GS) was cloned on a recombinant plasmid pGS4 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The nucleotide sequence of a 2423 bp DNA segment containing the GS-coding region of B. fibrisolvens was determined and the complete amino acid sequence (701 residues) was deduced. Comparisons of the derived B. fibrisolvens GS protein sequence with the amino acid sequences of GS from other bacteria indicate that it is the second reported example of a type III GS, originally identified in the obligate anaerobe Bacteroides fragilis. The presence of GS in B. fibrisolvens cells and the regulation of the cloned GS in E. coli cells was demonstrated by Western blot analysis.
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The use of bacterial luciferase genes as reporter genes in Lactococcus: regulation of the Lactococcus lactis subsp. lactis lactose genes
More LessSummary: Lactose metabolism is an important industrial trait in dairy lactococci. In Lactococcus lactis, lactose is taken up via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and is subsequently metabolized via the glycolytic and tagatose 6-phosphate pathways. Genes for the lactose-specific PEP-PTS proteins, phospho-β-galactosidase and tagatose 6-phosphate pathway enzymes are encoded by a single 8 kb operon, lac ABCDFEGX, and there is a divergently transcribed lacR repressor gene. Transcriptional fusions of both the lac operon promoter and the lacR promoter to the luxAB genes of Vibrio fischeri were used to investigate the regulation of expression of both promoters. In vivo bioluminescence assays demonstrated that lacR negatively regulates the lac operon and also autoregulates itself. Induction of transcription occurred for both promoters during growth on lactose: sevenfold for lacR and fivefold for the lac operon. The lacR promoter was demonstrated to be a particularly strong promoter, being approximately four times more efficient than the lac operon promoter. Both promoters provide good potential for the inducible expression of foreign proteins in Lactococcus.
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Association of the lactococcin A immunity factor with the cell membrane: purification and characterization of the immunity factor
More LessSummary: The physicochemical characteristics of the lactococcin A immunity protein, as deduced from its gene sequence, were used to devise a procedure for its purification. The protein was purified from cell extracts by cation-exchange and reverse-phase chromatography. As judged from the amino acid composition and amino acid sequencing, the immunity protein is not post-translationally processed by cleavage at its N- or C-terminus. Consequently, the absorption coefficient at 280 nm, the isoelectric point, and the molecular mass of the immunity protein may be calculated to be, respectively, 8·2 × 103 M−1 cm−1, 10·2 and 11163 Da from the amino acid sequence predicted from the nucleotide sequence. The immunity protein is a major cell protein component – one cell may contain (to an order of magnitude) 105 molecules – and it is in part associated with the cell membrane, as judged by immunoblot analysis of membrane vesicle-associated proteins. Exposing lactococcin-A-sensitive cells to an excess of the immunity protein did not affect the lactococcin-A-induced killing of the cells, indicating that the immunity protein does not protect cells by simply binding to lactococcin A, nor to externally exposed domains on the cell surface. Exposing immune-positive cells to antiserum against the immune protein did not sensitize the cells to lactococcin A, suggesting that the immunity protein in fact does not act extracellularly.
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Identification of the L-tartrate dehydratase genes (ttdA and ttdB) of Escherichia coli and evolutionary relationship with the Class I fumarase genes
More LessSummary: The genes encoding an oxygen-labile stereospecific L-tartrate dehydratase (L-Ttd, EC 4.2.1.32) have been identified as the orfZ1 and orfZ2 genes located upstream of the rpsU-dnaG-rpoD operon at 67 min in the Escherichia coli linkage map. They were previously cloned and sequenced by M. Nesin and others (Gene 51, 149-161, 1987) and have now been independently cloned, partially resequenced, and designated as an operon (ttdAB) containing two translationally coupled genes. The enzyme behaves as a tetramer (Mr 105000) containing two pairs of non-identical subunits, TtdA (Mr 32589) and TtdB (Mr 22641), which otherwise resembles the homodimerie iron-sulphur-containing Class I fumarases of E. coli and Bacillus stearothermophilus. The amino acid sequences of the TtdA-TtdB subunits are colinearly related to a single fumarase subunit, indicating a common evolutionary ancestry. E. coli can use L-, D- and meso-tsrtrates as aerobic growth substrates and as reducible substrates for supporting anaerobic growth on glycerol. L-Ttd was induced during anaerobic growth on glycerol plus L- and meso -tartrates, and a stereospecific D-tartrate dehydratase was induced by all three stereoisomers under comparable conditions. No meso-tartrate dehydratase was detected, nor were any dehydratases detected after aerobic growth on tartrate minimal media suggesting that different catabolic routes operate under aerobic conditions.
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Analysis of heterokaryon incompatibility between heterokaryon-compatibility (h-c) groups R and GL provides evidence that at least eight het loci control somatic incompatibility in Aspergillus nidulans
More LessSummary: Protoplast fusion was used to hybridize strains 99-6 (h-cR) and 7-141 (h-cGL) in the Birmingham collection of Aspergillus nidulans. A sparsely conidiating, brown-pigmented diploid strain (MA7) was isolated. Inocula from MA7 were haploidized on medium containing Benlate and a haploid progeny sample collected. Heterokaryon compatibility testing of selected progeny strains assayed each linkage group in turn and established that h-cR and h-cGL were hetero-allelic for heterokaryon incompatibility (het) loci located on linkage groups III and V. Two selected MA7 progeny strains were sexually crossed to an h-cGL strain. Compatibility classification of the progenies of these crosses indicated that three het genes were located on linkage group III and two on linkage group V. Combination of these results with previous work using h-c groups A, B and Q in comparison with h-cGL indicates that a minimum number of eight and a maximum number of 18 het loci, spread over five linkage groups, are necessary to explain the compatibility relationships among these five h-c groups.
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- Immunology
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Immunological and functional characterization of proteins of the Mycobacterium tuberculosis antigen 85 complex using synthetic peptides
More LessSummary: As tuberculosis re-emerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabeled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis.
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Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115
More LessSummary: Twenty-two monoclonal antibodies (mAbs) specific for smooth lipopolysaccharide (S-LPS) were generated by fusion of spleen cells from mice infected with the rough Brucella melitensis strain B115 with the NSO myeloma. According to reactivity in enzyme-linked immunosorbent assay (ELISA) with O-polysaccharide (O-PS) and absence of reactivity with rough lipopolysaccharide (R-LPS), it was postulated that these mAbs recognized epitopes present on the O-PS. Most of the mAbs reacted equally well in ELISA and immunoblotting with S-LPS types of Brucella A and M dominant strains and were designated as specific for common (C) epitopes. Three mAbs were highly specific for M dominant S-LPS. All these mAbs, in contrast to a mAb specific for the A epitope, showed little or no cross-reactivity with Yersinia enterocolitica O:9 S-LPS. S-LPS of B. melitensis B115 was extracted and analysed by immunoblotting and ELISA with mAbs specific for A, M and C epitopes. Reactivity of the mAbs with this S-LPS was compared to reactivity with S-LPS of A and M dominant smooth Brucella strains. The results suggest that S-LPS of B. melitensis B115 bears mainly C epitopes and a few M epitopes. The very weak reactivity of this S-LPS with the mAb specific for the A epitope and the fact that the mAbs specific for C and M epitopes showed little or no cross-reactivity with Y. enterocolitica O:9 S-LPS suggest that O-PS from this rough strain could be used to distinguish Y. enterocolitica O:9 infection from Brucella infection. The potential value of the rough B. melitensis strain B115 as a vaccine strain is also discussed.
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The production and partial characterization of a monoclonal IgG antibody specific for moulds belonging to the order Mucorales
Summary: A monoclonal antibody (mAb) was raised against extracellular polysaccharides from Mucor racemosus after intrasplenic immunization of mice. An indirect ELISA and a dot-blot assay were developed with this mAb. The IgG antibody was found to be very specific for all mould species tested belonging to the order of Mucorales, except species belonging to the genus Mortierella sensu stricto. No cross-reactions were observed with other moulds or yeasts. The immunoreactivity of the polysaccharides of these moulds with this mAb is based on carbohydrate epitopes, in which fucose residues probably play an important role. The mAb may be suited for specific detection of species of the genera Mucor, Rhizopus, Rhizomucor, Thamnidium, Absidia, Syncephalastrum and species belonging to the Mortierella isabellina group in food, and possibly for diagnosis of mucormycosis in humans.
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- Pathogenicity And Medical Microbiology
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Cloning, DNA nucleotide sequence and distribution of the gene encoding the SEF14 fimbrial antigen of Salmonella entevitidis
More LessSummary: Monoclonal antibody 69/25, specific for the Salmonella entevitidis fimbrial antigen (SEF14), was used to screen a pUC-based S. enteritidis gene library and a positive clone was identified. Subcloning experiments demonstrated that a 584 bp DraI DNA fragment was the minimal chromosomal segment capable of directing SEF14 antigen expression. Western blotting of Escherichia coli recombinants identified a gene product of Mr 16000 as a precursor to the M r 14300 mature fimbrial subunit protein. The DNA nucleotide sequence of the DraI fragment was determined and was shown to contain a single open reading frame with two potential f-Met start codons and a hydrophobic signal sequence. Downstream of a putative peptidase cleavage site, the deduced amino acid sequence showed considerable homology with the N-terminal amino acid sequence of what was originally described as the type 1 fimbrial subunit of Salmonella enteritidis and later redefined as SEF14. The gene encoding SEF14, designated as sefA, was shown to be limited in distribution to Salmonella blegdam, S. dublin, S. enteritidis, S. gallinarum, S. moscow, S. pullorum, S. rostock, S. seremban and S. typhi, all belonging to Salmonella group D. However, expression of the SEF14 antigen was limited to S. dublin, S. enteritidis, S. moscow and S. blegdam. The nucleotide sequence of the sefA gene shared no homology with the Salmonella fimA gene encoding type 1 fimbriae, and these genes showed distinct patterns of distribution within salmonellae.
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Sequence of the gtfK gene of Streptococcus salivarius ATCC 25975 and evolution of the gtf genes of oral streptococci
More LessSummary: Many strains of oral streptococci secrete glucosyltransferases (GTFs) that polymerize sucrose into glucans that form an integral part of the plaque matrix on the tooth surface. Recently, we reported the cloning of two closely linked GTF-encoding genes (gtfJ and gtfK) from Streptococcus salivarius ATCC 25975 as well as the sequence of gtfJ, which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtfK, which encodes a primer-dependent GTF that synthesizes a soluble product (a GTF-S), as well as the sequence of a small downstream open reading frame of unknown function. The deduced sequence of GtfK was compared with those of seven other streptococcal Gtfs and an unrooted phylogenetic tree constructed. This analysis suggested that Gtfs with similar product specificities do not form phylogenetic clusters and was consistent with currently accepted phylogenetic schemes. The tree was tested by constructing a series of “sub-trees” from different blocks of the alignment. Evidence was obtained for recombination events involving gtfB and gtfC from S. mutans GS-5, gtfJ and gtfK from S. salivarius, as well as the gtfI genes from S. downei and S. sobrinus. The recombination events between gtfB and gtfC, and between the two gtfI genes, were confirmed by examining divergences at silent sites.
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Rapid, amplification-based fingerprinting of Mycobacterium tuberculosis
Summary: Insertion element IS6110 occurs in multiple copies throughout the Mycobacterium tuberculosis genome, and the variability of its insertion sites is the basis for the IS6110 restriction fragment length polymorphism (RFLP) method for typing. We describe a novel gene amplification method to assess the variability of the location of IS6110. A unilateral-nested polymerase chain reaction and hybridization procedure was used to measure the variability in the distances between IS6110 elements and copies of a major polymorphic tandem repeat sequence of M. tuberculosis. The pattern of amplicons produced could be used to cluster epidemiologically related strains of M. tuberculosis into groups which correlated with the groups formed using IS6110-RFLP typing. Reliable patterns can be generated directly from sputum specimens as well as from M. tuberculosis cultures. We designated the novel method as IS6110-ampliprinting.
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Mapping of a surface-exposed B-cell epitope to the variable sequent 3 of the major outer-membrane protein of Chlamydia trachomatis
More LessSummary: A B-cell epitope, AEFPLDIT, was located to the variable sequent 3 of the major outer-membrane protein (MOMP) using the monoclonal antibody L3-1, raised to the Chlamydia trachomatis serovar L3 MOMP. By Western blot and inclusion immunofluorescence assay the monoclonal antibody recognized all the C complex and C-related complex serovars of C. trachomatis, except serovar C. Dot-blot and ELISA data using native elementary bodies indicated that the epitope was surface exposed. The monoclonal antibody, at concentrations of 10 and 100 μg per 107 chlamydial inclusion-forming units, was able to neutralize the infectivity of chlamydia in an in vivo assay but did not neutralize chlamydia in vitro or in a mouse toxicity assay. A peptide corresponding to the variable sequent 3 has previously been shown to also elicit a T-cell response; thus, careful consideration should be given to inclusion of this region of the major outer-membrane protein in a subunit vaccine.
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Stimuli that induce production of Candida albicans extracellular aspartyl proteinase
More LessSummary: Several species of the opportunistic fungal pathogen Candida produce an extracellular aspartyl proteinase that may assist the organism to invade and colonize host tissues, evade the host immune response and assimilate nitrogen from proteinaceous sources. Although addition of exogenous proteins, such as bovine serum albumin (BSA), to cultures of C. albicans is known to elicit proteinase production, the precise molecular mechanisms controlling regulation of proteinase induction are unknown. We have examined the ability of a variety of macromolecules to induce proteinase production using a chemically-defined nitrogen-limited growth medium and a rapid, sensitive microtitre fluorescent assay for proteinase activity in culture supernatants. BSA and the extracellular matrix protein collagen induced proteinase production. Homopolymers of both poly-L- and poly-D-glutamate also induced proteinase activity, whereas polyglycine, heparin sulphate and dextran sulphate did not. Thus, molecular recognition of proteinase-inducing stimuli is not highly stereospecific, but apparently requires both main- and side-chain interactions. Peptides 8 or more residues in length generally induced proteinase production while most shorter peptides did not. These data reveal that internalization of small peptides with less than 7 residues by peptide transport was not the inducing signal for proteinase production, since Candida dipeptide and oligopeptide permeases do not efficiently transport peptides of more than 6-7 residues. In addition a tight-binding synthetic inhibitor of Candida proteinase (K i = 0.17 nM) prevented growth of C. albicans on BSA as a sole nitrogen source by blocking protein degradation. Immunodetection of proteinase in these culture supernatants suggests that fully intact proteins, in addition to peptide fragments of sufficient size, are capable of inducing proteinase production. A model involving stimulation of a plasma membrane signal transduction event by extracellular protein and/or polypeptide ligands of more than seven residues is compatible with these data.
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- Physiology And Growth
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Response of catalase activity and membrane fluidity of aerobically grown Schizosaccharomyces pombe and Saccharomyces cerevisiae to aeration and, the presence of substrates
More LessSummary: Intracellular catalase (EC 1.11.1.6) activity of permeabilized aerobically grown cells of Schizosaccharomyces pombe was insensitive to cell aeration and inhibition of protein synthesis, and was only mildly enhanced by the presence of glucose and ethanol via de novo protein synthesis. By contrast, the intracellular catalase activity of Saccharomyces cerevisiae, which, in freshly harvested cells, was two to three times lower than that in Sch. pombe, increased on aeration without substrates or with ethanol and was inhibited on aeration with glucose following cell permeabilization. The enhanced intracellular activity was due to de novo protein synthesis while the inhibitory effect of glucose, absent in Sch. pombe, was caused by one of the major glucose metabolites, succinate. The intact-cell catalase activity of both yeasts increased greatly during aeration. In Sacch. cerevisiae, this increase was again prevented by glucose. In parallel, export of catalase to the cell surface increased in both yeasts. This was especially conspicuous in Sch. pombe aerated in the presence of ethanol, and may represent a protective mechanism against the damaging effects of ethanol. The cell-surface-bound catalase activity was confirmed in isolated plasma membranes of both yeasts. The fluidity of the plasma membrane increased during aeration. This effect was further stimulated by the presence of glucose and to a lesser extent by ethanol. Both yeasts exhibited increased extracellular catalase activity during aeration which could not be caused entirely by cell lysis. In Sch. pombe this activity was strongly enhanced by the presence of ethanol.
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Fatty acid composition and molecular order of phospholipids from Euvotium chevalieri in response to changes in water activity
More LessSummary: The mycelial growth of Eurotium chevalieri was examined at different water activities (a w) using glycerol as the osmoticum. Growth was optimal at 0.90 a w and restricted at 0.995 a w highlighting the xerophilic behaviour of E. chevalieri. Decreased a w produced an increase in the proportion of oleic acid (C18:1) at the expense of the proportion of linoleic acid (C18:2) of cellular phospholipids. The degree of unsaturation of phospholipid fatty acids showed a 20 % decrease between 0.995 and 0.80 a w of growth. Steady-state fluorescence anisotropy (r s) and fluorescence lifetime (τ) measurements for liposomes prepared from cellular phospholipids of E. chevalieri and labelled with DPH (1,6-diphenyl-1,3,5-hexatriene) were made at 25 °C. The lipid order parameter (S, describing molecular order) and the rotational correlation time (ϕ, describing molecular dynamics) were calculated from r s and τ data. Except at 0.995 a w, a decrease in a w was accompanied by increasing r s and S values, indicating a rigidification of membranes, while ϕ values were not significantly different. Plots of order parameters and their first derivatives as a function of temperature exhibited break areas in the temperature range 20-48 °G. These large temperature ranges for lipid transitions could correspond to chain melting of complex lipid systems which made up the liposomes prepared from phospholipids of E. chevalieri. However, as a w decreased, the transition temperatures increased globally, between 0.97 and 0.90 a w.
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- Plant-Microbe Interactions
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Production and regulation of potato-scab-inducing phytotoxins by Streptomyces scabies
More LessSummary: Phytotoxins with potato-scab-inducing activity were produced by the pathogenic Streptomyces scabies strain RB2 when grown on oatmeal agar medium or in oatmeal broth medium. Five compounds isolated from cell filtrates were identified as thaxtomin compounds 1-5, previously reported as produced by S. scabies grown on potato slices. The optimum temperature for phytotoxin production in oatmeal broth medium was 28 °C. Production of thaxtomin A, the major product, was repressed at least 130-fold when S. scabies RB2 was grown in oatmeal broth medium supplemented with 0.5% glucose. Thaxtomin A production was also repressed by tryptophan and tyrosine, precursors of which may be involved in feedback inhibition of early steps in biosynthesis. Phytotoxins were secreted by the organism when the cells reached late exponential to early stationary phases of culture growth. The time during growth at which each thaxtomin compound was produced suggests a pathway for the latter steps in thaxtomin biosynthesis.
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Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction
More LessSummary: The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.
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- Systematics
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phytogeny and phenotypic characterization of the stalk-forming and iron-oxidizing bacterium Gallionella ferruginea
More LessSummary: The 16S rRNA gene of Gallionella feiruginea was amplified by polymerase chain reaction and sequenced by direct double-stranded sequencing. The phylogenetic analysis placed G. feiruginea in the -group of the Proteobacteria, with 90.0% similarity to Nitrosolobus multiformis and 88.6% to Rhodocyclus purpureas. The published phenotypic characteristics of G. ferruginea were compiled and supplemented with growth experiments using ferrous iron, thiosulphate and sulphide as electron donor, and nitrate as nitrogen source. G. ferruginea is a Gram-negative, curved bacterium with one polar fiagellum. It grows auto- and mixotrophically with CO2, glucose, fructose and sucrose as carbon sources, ferrous iron as an electron donor and ammonium or nitrate as nitrogen sources. Two G. feiruginea specific oligonucleotide probes are suggested. An iron-oxidizing bacterium without stalk-forming ability, but with the same growth pattern as G. ferruginea, was identified as G. ferruginea by comparison of highly variable parts of the 16S rRNA gene. This indicates that the stalk is not essential for growth.
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- Genome Analysis
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Organization of the Escherichia coli and Salmonella typhimurium chromosomes between flagellar regions IIIa and IIIb, including a large non-coding region
More LessSummary: Flagellar regions IIIa and IIIb of the Escherichia coli and Salmonella typhimurium chromosomes (at 40 min and 42-43 min, respectively) has been shown to be separated by DNA unrelated to flagellar function, with region IIIa being immediately followed by a gene, amyA, that encodes a cytoplasmic α-amylase. The chromosome between amy A and flagellar region IIIb has now been investigated. The high level of DNA similarity between the E. coli and S. typhimurium sequences that exists in flagellar region IIIa and in amy A continues initially, with three genes of unknown function; in E. coli, there may be a fourth gene. The remainder of the region, up to the start of flagellar region IIIb, lacks any obvious open reading frames, scores poorly on an algorithm for coding probability, has a high A + T content, and is totally dissimilar in the two species. We conclude that it is non-coding. In E. coli this region extends for 2 7 kb and in S. typhimurium for 08 kb. These values are unusually large for prokaryotes, where the non-coding regions between operons are generally quite short. The data, which are discussed in the context of a hypothesized disruption of a contiguous ancestral flagellar region, may give new insight into the organization and evolution of the bacterial chromosome.
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Clonal size-variation of rDNA cluster region on chromosome XII of Sacchavomyces cerevisiae
More LessSummary: Using pulsed-field gel electrophoresis (PFGE), we have demonstrated clonal variation in the size of chromosome XII in a diploid strain of Sacchavomyces cerevisiae X2180-2D. The sizes of the two chromosome XII homologues were very different: 2600 (L-type) and 1450 kb (S-type). The frequency with which we detected clonal size variation in the diploid, compared to that of the parental clones, was about 15-50% of the progeny clones and the range of the size variation of the homologues was 2580-2680 kb (L-type) and 1340-1500 kb (S-type), respectively. The homologue of the L-type appeared to be more frequently variable than that of the S-type. The size variation was shown to be derived from size changes in the rDNA cluster region, which is present in chromosome XII, by digesting the chromosome with XhoI, whose cutting site is not present in a rDNA repeat unit, and hybridizing to rDNA probes. The clonal size variation was also investigated in haploids from spores after meiosis. The L-type and S-type chromosomes segregated 2:2 in an ascus and the sizes of all the S-type chromosomes were shifted up, compared to the original diploid, though the L-type ones were stable. The S-type sizes of 1340, 1450 and 1780 kb in the original diploids changed into the ranges of 1475-1610 kb, 1520-1680 kb and 1820-2010 kb, respectively, in the segregants. Furthermore, we observed that the size of S-type chromosomes in haploid cells was gradually increasing in mitosis during successive subcultures. The rDNA units appeared to be amplified on the S-type chromosome.
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- Instructions To Authors
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