- Volume 140, Issue 9, 1994
Volume 140, Issue 9, 1994
- Sgm Special Lecture
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- Microbiology Comment
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- Antigens And Immunity
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Specific and cross-reacting monoclonal antibodies to Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides
More LessThree groups of monoclonal antibodies (mAbs) were produced that would be useful for immunochemical typing and diagnosis of infections due to Bordetella species, and for the structural analysis of their lipopolysaccharides. PP6, a representative of the first group, recognizes an epitope shared by smooth-type Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides (LPS). This epitope is carried by structurally identical polymeric O-chains (POC) present on both LPS molecules. PP8 and PP9 are representatives of the second group of mAbs. The interaction of PP8 and PP9 with B. parapertussis and B. bronchiseptica LPS requires POC, but periodate-sensitive sugar units of the core are also involved in the binding. The mAb BRg1 belongs to the third group, and specifically recognizes B. bronchiseptica LPS. Binding and inhibition studies with various Bordetella LPS molecules, and with their polysaccharide fragments, indicated that BRg1 interacts with a structure located at the hinge between the POC and a core region of the B. bronchiseptica LPS containing periodate-resistant sugars. This suggests that the structures of the hinge regions of the B. parapertussis and B. bronchiseptica LPS are different.
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- Biochemistry
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Characterization of chitin synthase from Botrytis cinerea
More LessChitin synthase in a microsomal preparation from Botrytis cinerea had an apparent K m for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent K i values of approximately 0.1 μM and 6 μM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (K iapp 54 μM). Preincubation of microsomes for 2 h at 25 °C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 μg ml-1) or cytosol (350 μg cytosolic protein ml-1) for 10 min at 25 °C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.
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Subcellular localization, abundance and stability of chitin synthetases 1 and 2 from Saccharomyces cerevisiae
More LessThe existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different localization. The subcellular distribution of chitin synthetases 1 and 2 (Chs1 and Chs2) was determined in cell-free extracts of Saccharomyces cerevisiae fractionated by sucrose density gradient sedimentation. Chs1 was examined in two strains: ATCC 26109, a wild-type strain, and D3C (MATα ura3-52). Chs2 was investigated in a strain (D3B) freed of Chs1 by gene disruption (MATa his4 ura3-52 chs1::URA3). A prolonged, strong centrifugation (20 h at 265000 g) was necessary to cleanly resolve two major populations of chitin synthetase particles: chitosomes (a population of microvesicles of low buoyant density, d = 1.15 g ml-1) and plasma membrane (a population of vesicles of high buoyant density, d = 1.21 g ml-1). Chs1 and Chs2 were both present in chitosomes and plasma membrane, but the relative distribution of each chitin synthetase in these two membranous populations varied. Chs2 was much less abundant than Chs1 and required Co2+rather than Mg2+as a cofactor. A salient finding was the high sensitivity of chitosomal Chs2 to high centrifugal forces. The subcellular distribution of 1,3-β-glucan synthetase was the same in the three strains studied, i.e. unaffected by the presence or absence of Chs1. Culture conditions affected the profiles of chitin and glucan synthetases: the relative abundance of Chs1 in chitosomes or plasma membrane was quite different in cells grown on two different media but the buoyant density was not affected; in contrast, there was shift in the buoyant density of the two peaks of 1,3-β-glucan synthetase. We concluded that the subcellular localization of Chs1 and Chs2 remains the same despite genetic and other differences in the properties of these enzymes. We confirmed that 1,3-β-glucan synthetase and chitin synthetase exhibit a partially different subcellular distribution-an indication that these two enzymes are mobilized through different secretory pathways.
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Characterization of (1,3)-β-glucan synthase in Candida albicans: microsomal assay from the yeast or mycelial morphological forms and a permeabilized whole-cell assay
More LessA systematic evaluation of the in vitro (1,3)-β-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5′-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 °C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 μg protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent K m for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 °C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis. This study represents the first characterization of glucan synthase using yeast and mycelial phase microsomes and adaptation of a permeabilized system using a single C. albicans strain with standardized assay conditions.
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The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by Gram-positive bacteria
More LessColicin V is a ribosomally synthesized antimicrobial peptide produced by Escherichia coli. Four recently characterized genes, arranged in two convergent operons on the plasmid pColV-K30, are required for colicin V synthesis, export and immunity. We report the purification and N-terminal amino acid sequencing of the colicin V protein. Our results demonstrate that the colicin V primary translation product, which consists of 103 amino acids, is proteolytically processed. A leader peptide, consisting of 15 amino acid residues, is removed from the N-terminus during maturation of colicin V. This leader peptide is not related to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the Sec pathway. The molecular mass of colicin V, obtained by mass spectrometry analysis, showed that the peptide consists of only unmodified amino acids. The deduced amino acid sequence of the leader peptide was highly homologous to the N-terminal extensions found in non-lantibiotic, peptide bacteriocins produced by Gram-positive bacteria. These findings strongly indicate that colicin V belongs to a family of small peptide bacteriocins that have been found previously only among the Gram-positive lactic acid bacteria.
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Evidence for a polysaccharide-binding domain in Hormoconis resinae glucoamylase P: effects of its proteolytic removal on substrate specificity and inhibition by β-cyclodextrin
More LessThe hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by β-cyclodextrin with apparent K i values of 190 μM, 13 μM and 1.4 μM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0.3 × K m and up to 4 mM β-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by β-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4.3 and 4 °C with an association constant of 3.4 × 105M-1. Sequence alignment studies showed raw-starch-binding consensus amino acids in the C-terminal part of glucoamylase P. Partial hydrolysis with papain resulted in degradation of deglycosylated glucoamylase P into three fragments of 53, 51 and 14 kDa, respectively, as estimated by SDS-PAGE. The amino-terminal sequences of the 51 and 53 kDa fragments were identical with that of native glucoamylase P. The amino terminus of the 14 kDa fragment (Ser-Ser-X-Gln-Val-Ser-), corresponded to the sequence starting at residue 474 of intact glucoamylase P. Kinetic measurements of truncated glucoamylase P showed changes in the K m values of larger polysaccharides, but no changes in K cat values compared to the intact enzyme. It was concluded that glucoamylase P contains a catalytic core domain and a raw-starch-binding domain involved in inhibition of polysaccharide hydrolysis by β-cyclodextrin.
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- Development And Structure
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The Neurospora crassa chs-2 gene encodes a non-essential chitin synthase
More LessChitin is a structural component of morphologically distinct structures assembled during various phases of growth and development in filamentous fungi. In Neurospora crassa, at least three different DNA fragments related to chitin synthase have been identified. In this study we cloned, sequenced and characterized the chitin synthase 2 structural gene (designated chs-2). The amino acid sequence deduced from the cloned chs-2 genomic DNA fragments is very similar to that of chitin synthase genes isolated from other fungi. Inactivation of the N. crassa chs-2 gene by repeat-induced point (RIP) mutation produced progeny which under standard growth conditions were indistinguishable from the wild-type. However, a significant reduction in chitin synthase activity and increased sensitivity to the phosphatidylcholine biosynthesis inhibitor edifenphos are characteristic of the chs-2 RIPstrain.
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Sexual agglutination substances require a ‘carrier’ glycoprotein for integration into the cell wall of Saccharomyces cerevisiae
More LessSexual agglutination, caused by agglutination substance (AS) on a and α cell walls, is the first indispensable step of the mating reaction in ascosporogenous yeasts including Saccharomyces cerevisiae. The AS biosynthetic process in S. cerevisiae was investigated by pulse label-chase experiments with analysis by polyacrylamide gel electrophoresis (PAGE) for 16 h in the presence of urea. Because of its low mobility, AS can be separated from other proteins by prolonged PAGE. Nascent AS was integrated into cell walls after it linked covalently to a ‘carrier’ glycoprotein. The results suggest that the ‘carrier’ is synthesized stepwise through three distinct precursors (III → II → I). The ‘carrier’ glycoprotein (I) and its precursors (II, III) were synthesized in both a, α haploid and a/α diploid cells. The N-glycosylation linkage inhibitor, tunicamycin, and protein synthesis inhibitor, puromycin, inhibited the III to I maturation. The results indicated that both the ‘carrier’ and the nascent active site of AS linked to the ‘carrier’ are integrated into the wall in a haploid cell while the ‘carrier’ alone is integrated in a diploid cell.
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Immunogold localization of GyrA and GyrB proteins in Escherichia coli
More LessImmunogold preparations of Escherichia coli, using anti-GyrA and anti-GyrB antibodies to the subunits of DNA gyrase, showed clear labelling with both secondary antibody and protein A-gold conjugates. Both proteins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid. This partitioning of gyrase proteins between nucleoid and cytoplasm was non-random and was consistently observed for a range of different cell preparations. Total gold particle counts were highly variable but suggested levels of at least 1000-3000 molecules per cell for both GyrA and GyrB. Sequential treatment with both anti-GyrA and anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two groups of molecules. Treatment of cells with chloramphenicol caused marked changes in nucleoid conformation, but no reduction in cytoplasmic labelling of gyrase proteins. On the assumption that gyrase complexes within the nucleoid are not differentially masked from the monoclonal antibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DNA, but are distributed randomly throughout the cytoplasm.
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- Environmental Microbiology
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Phenotypic and genotypic diversity of fluorescent pseudomonads isolated from field-grown sugar beet
More LessA sample of 30 fluorescent pseudomonads isolated from the phyllosphere of sugar beet throughout a single growing season and shown to be closely related on the basis of fatty acid methyl ester (FAME) analysis was subjected to detailed phenotypic and genotypic characterization. Phenotypic traits were assessed on the basis of biochemical properties, assimilation of sole carbon sources, FAME analysis, organic pyrolysate content (MS-pyrolysis), and total cellular protein profiles. With the exception of total cellular protein profiles, numerical analysis of the data revealed two main clusters, each of which was divided into several subclusters. Numerical analysis of total cellular protein data failed to differentiate isolates into two main clusters, but nevertheless grouped isolates into six subclusters. On the basis of biochemical and carbon source assimilation profiles, 19 isolates were identified as Pseudomonas fluorescens biovar V, eight isolates as P. fluorescens biovar III and three isolates as P. syringae pathovar syringae. In general, all methods of phenotypic analysis grouped isolates according to time of sampling and leaf type. Genome analysis was undertaken by pulsed-field gel electrophoresis (PFGE) of Pacl, Spel, Swal and Xbal macrorestriction fragments and revealed the presence of eight distinct genomic (clonal) groups. These groups correlated closely with the clusters generated by numerical analysis of phenotypic data, but there was no correlation between macrorestriction fragment profile and isolate identification; in fact the variation in macrorestriction fragment patterns within P. fluorescens biovars was as great as the variation detected between biovars, and between P. fluorescens and P. syringae. Statistical evaluation of macrorestriction fragment patterns revealed two examples of recent strain divergence: one was due to the presence of a 400 kbp plasmid within one isolate of a collection of nine otherwise genomically identical isolates, and the other was observed between two phenotypically similar isolates sampled 220 d apart. Genetic variation was expressed in terms of nucleotide diversity (π) and pairwise comparisons yielded values ranging from 0.0029 to 0.1517. The mean intrapopulation genetic variation was high (0.0993), but limited genetic variation was detected among isolates sampled on each occasion. Taken together this suggests a population comprised of a variety of apparently distantly related clones (genomic groups), each adapted to local conditions. Genome sizes were estimated from the sum of Spel restriction fragments and ranged from 4.2 to 5.5 Mbp. Examination of the distribution of Xbal, Spel, Swal and Pacl restriction endonuclease sites showed that the distribution of Spel sites differed significantly from the expected (random) distribution.
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The identification of Bradyrhizobium japonicum strains isolated from Italian soils
Pyrolysis mass spectrometry (PYMS) and PCR using arbitrary primers were used to characterize strains of Bradyrhizobium japonicum isolated from fields in northern Italy. The combination of techniques allowed us to identify bacteria derived from inoculants and to demonstrate that information about the nature of one inoculant was incorrect. PYMS also indicated that the derivatives from one inoculant formed two distinct populations: one like the parent strain and the other altered phenotypically.
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- Genetics And Molecular Biology
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Temporal transcription map of the Lactococcus lactis bacteriophage sk1
More LessBacteriophage sk1 is a small isometric-headed lytic phage that infects Lactococcus lactis. The phage has a linear double-stranded DNA genome of 28 kbp, with cohesive ends. RNA was prepared from phage-infected L. lactis cells harvested at various intervals after infection, and the RNA molecules were resolved by E1ectrophoresis. Northern blots of these gels were hybridized with sk1 DNA probes and the results obtained from these experiments, together with the results of primer extension analyses, enabled a transcription map of the phage genome to be prepared. Three classes of phage transcripts, designated as early, middle or late based on their time of appearance, were detected. Seven partially overlapping early transcripts were detected; these were transcribed from a 10 kbp region of the phage. The nine middle transcripts were derived from a 2 kbp region, limited by cos at one end and the start of the early transcripts at the other. The early and middle transcripts were transcribed divergently from a region mapping at 26 kbp on the sk1 physical map. The four late transcripts were derived from a 16 kbp region of the phage limited at one end by cos. The late transcripts were transcribed in the opposite direction to the early transcripts and three of the late transcripts terminated in the same region of the phage genome as three of the early transcripts.
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Nucleotide sequence and structural organization of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533
More LessThe nucleotide sequence of the Leuconostoc lactis 533 cryptic plasmid pCI411 (2926 bp) was determined. Analysis revealed the presence of three open reading frames (ORFs). ORF 1 was capable of encoding a 24.9 kDa peptide which shared homology with the replication initiation protein (RepB) from a number of Gram-positive rolling circle plasmids. ORF 2 could encode a peptide of 6.6 kDa which was homologous to the RepC protein of the lactococcal plasmid pWV01. A function could not be assigned to ORF 3, which was capable of encoding a 12.1 kDa peptide. Transcription—translation analysis indicated the presence of three peptides of the predicted molecular masses. A putative double strand origin of replication (DSO) was identified which showed strong similarity with the DSO of a number of Gram-positive plasmids including pE194 from Staphylococcus. Structural analysis identified a number of direct and indirect repeats in addition to putative recombination-specific sites (RSA and RSB) in the non-coding region of pCI411. The observed characteristics suggest that this plasmid replicates using the rolling circle mechanism. pCI411, which could be introduced into Leuconostoc, Lactococcus, Streptococcus, Lactobacillus and Bacillus is the first plasmid from the genus Leuconostoc to be characterized in such detail.
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The 387 kb linear plasmid pPZG101 of Streptomyces rimosus and its interactions with the chromosome
More LessThe linear plasmid pPZG101 of Streptomyces rimosus R6 was restriction mapped with the enzymes Asel, Bfrl, Dral and Xbal. It is 387 kb in size and the ends are inverted repeats of at least 95 kb in length. Twenty spontaneous morphological variants and seventeen auxotrophic mutants were screened for changes in the plasmid. Two strains were found that had lost all plasmid sequences. Four strains had integrated parts of the plasmid into the chromosome. Restriction analysis suggested that at least three of the integrated strains had retained free plasmid ends. If it is assumed that the chromosome of S. rimosus R6 is linear, this might be explained by replacement of one or both chromosome ends by a plasmid end. One strain, which overproduced oxytetracycline, carried an enlarged linear plasmid of 1 Mb in size that had acquired chromosomal sequences from the oxytetracycline biosynthesis cluster.
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Analysis of Bacillus subtilis tag gene expression using transcriptional fusions
More LessFive of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.
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Identification of a control region for expression of the forespore-specific Bacillus subtilis locus spoVA
More LessThe role of a 20 bp conserved region located 45-64 nucleotides 5′ of the spoVA transcription start point in Bacillus subtilis and Bacillus licheniformis was investigated by deletion analysis and by mobility shift assay. Deletions 5′ of this conserved sequence had little effect on expression of a spoVA-lacZ fusion, whereas deletions extending into the sequence reduced expression of the spoVA-lacZ fusion by 85%. The timing of expression of spoVA was not affected by deletion of the sequence. The region was shown by mobility shift assays to bind specifically to a protein. Binding activity was detected in protein extracts prepared from bacteria 1 h or more after they had started to sporulate, but not in extracts prepared from vegetative bacteria. Mutations in all known spoO loci were screened but none prevented appearance of the binding activity; nor did mutations in any of the stage II and III loci tested. It is concluded that the 20 bp conserved region is the binding site of an activator that is subject to temporal regulation independent of known spo loci.
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Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme
The molecular characteristics of the ethylene-forming enzymes of strains of Pseudomonas syringae were tested. The ethylene-producing activities of the nine strains as measured in vivo and in vitro were similar, except for that of P. syringae pv. mori M5. A polyclonal antibody and a DNA probe for the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 were prepared to investigate homologies among the proteins and genes for the ethylene-forming enzymes. With the exception of P. syringae pv. mori M5, eight strains tested expressed the same antigen as the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 and were homologous to DNA sequences on indigenous plasmids. Molecular masses of antigenic proteins from all ethylene-producing strains were 40 kDa. The N-terminal amino acid sequence of the purified ethylene-forming enzyme from P. syringae pv. glycinea KN130 was identical to that of the enzyme from P. syringae pv. phaseolicola PK2. These results show that the ethylene-forming enzymes encoded by the indigenous plasmid(s) in the pathogenic bacteria examined were similar.
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Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria
More LessWe have characterized a new family of proteins (the ROK family) which includes six transcriptional repressors for sugar catabolic operons, three sugar kinases, and three unidentified open reading frames. Analyses of the aligned sequences and phylogenetic tree construction allow predictions regarding the functional nature of conserved domains and residues within these proteins as well as the pathway of evolutionary divergence that gave rise to the family.
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Removal of the periplasmic DNase before electroporation enhances efficiency of transformation in the marine bacterium Vibrio alginolyticus
More LessWe have established a reliable procedure for electroporation in the marine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replicon were found to be stably maintained in the Vibrio cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mg2+in order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm-1. Under optimal conditions, about 105transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.
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Isolation of an Ustilago maydis gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase and expression of a C-terminal-truncated form in Escherichia coli
More LessA gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was isolated from the maize fungal pathogen Ustilago maydis. This was accomplished by identifying cDNA and genomic clones that hybridized to an internal fragment of the gene, amplified from U. maydis genomic DNA by PCR. The nature of the gene was determined by nucleotide sequence analysis, and by comparing the derived amino acid sequence of the gene with HMG-CoA reductases from yeast, and from other organisms. The hydrophobic nature of the N-terminal region of the deduced protein sequence also supported the view that this gene encoded HMG-CoA reductase. A C-terminal-truncated fragment of the U. maydis HMG-CoA reductase gene was shown to be expressed in Escherichia coli in a catalytically active form. The expressed protein was also shown to be sensitive to an inhibitor of mammalian HMG-CoA reductase activity.
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- Pathogenicity And Medical Microbiology
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Characterization of O-glycan moieties of the 210 and 240 kDa pig intestinal receptors for Escherichia coli K88ac fimbriae
More LessThe porcine brush border glycoproteins of 210 and 240 kDa, recognized by Escherichia coli K88ac fimbriae, contained O-linked oligosaccharides. The carbohydrate moieties were analysed by deglycosylation, lectin-binding and agglutination assays. Neuraminidase susceptibility of the 210 kDa receptor suggested that a sialoglycoprotein may act as receptor for the K88ac fimbriae. In contrast, K88ac-binding to the 210 and 240 kDa glycoproteins totally disappeared after fucosidase treatment, indicating the critical role of fucosyl residues at the receptor sites. Among the oligosaccharides extracted from these O-glycoproteins, K88ac fimbriae showed affinity for neutral sugar chains while sialylated species were not recognized. Our data suggest a possible role of the polypeptide backbone in the definition of receptor sites. Specific agglutination by K88ac-fimbriated E. coli of the erythrocytes of the hamster Mesocricetus auratus was inhibited by the anti-t peanut lectin and the lectins of Datura stramonium, Aleuria aurantia and Maackia amurensis. Hence, we propose that Galβ1-3GalNAc- and Fucα1-2Galβ1-3/4GIcNAc- are the main sequences mediating K88ac fimbrial binding. These structures were not detected in the non-adhesive piglet brush borders characterized by a high carbohydrate content. Additional oligosaccharides probably masked the underlying receptor structures.
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Hydrocortisone-enhanced growth of Aspergillus spp.: implications for pathogenesis
More LessAspergillus fumigatus and Aspergillus flavus are the most common cause of invasive mould infections worldwide and carry a high mortality. Corticosteroid therapy and Cushing's disease are associated with an increase in invasive aspergillosis. Corticosteroids impair immune function in mammals and, specifically, the conidicidal activity of human macrophages, which was thought to be sufficient explanation for this increased risk. However, we have found a 30-40% increase in growth rate of A. fumigatus and A. flavus exposed to pharmacological doses of hydrocortisone (a human glucocorticoid), suggesting an alternative or additional mechanism for the association. No significant effect was observed with other human steroids such as testosterone, oestradiol or progesterone, though a smaller (21%) but significant growth rate increase was obtained with the fungal sterol ergosterol. The presence of a ligand/receptor system is therefore possible in pathogenic Aspergillus spp. Although corticosterone-binding proteins have been identified in some yeast species, a demonstrable physiological effect has been lacking. Interruption of the putative ligand/receptor interaction could have a major effect on the growth and pathogenicity of A. fumigatus, providing opportunities for the development of alternative therapeutic strategies to those currently available.
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Determination of neutralizing epitopes in variable domains I and IV of the major outer-membrane protein from Chlamydia trachomatis serovar K
More LessChlamydia trachomatis is a leading cause of sexually transmitted diseases and a number of strategies have been developed to produce vaccines to prevent its transmission. The purpose of this study was to map the neutralizing epitopes of C. trachomatis major outer-membrane protein (MOMP) serovar K by using anti-MOMP antibodies and synthetic peptides. Seven anti-MOMP monoclonal antibodies and three polyclonal antisera were produced and characterized. Their fine specificity was defined by direct binding assay on 15 peptides of 10 amino acid residues, overlapping by five residues, corresponding to the four variable domains (VDI-VDIV: residues 64-85, 139-160, 224-237 and 287-319) of MOMP serovar K. Our data confirmed that a neutralizing epitope is found in VDIV, defined by peptides K12 and K13. This epitope is 296TTLNPTIAG304, which has never been reported as a neutralizing epitope of serovar K. Another neutralizing epitope, defined by peptide K2, has been identified in VDI. This epitope is in the same position as 71VAGLEK76, a peptide with neutralizing activity found in serovar A, but they are not identical because antibodies against peptide K2 do not bind to this epitope. No neutralizing epitope was found in the two other variable domains (VDII and III). In summary, two neutralizing sites, one in variable domain I and one in variable domain IV, were identified in serovar K.
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Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis: comparison of metronidazole-resistant and sensitive strains
More LessThe sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 μM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 μM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.
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- Physiology And Growth
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Alcohols lower the threshold temperature for the maximal activation of a heat shock expression vector in the yeast Saccharomyces cerevisiae
More LessWhen the yeast Saccharomyces cerevisiae is exposed to elevated growth temperatures, genes containing a heat shock element (HSE) in their promoters are activated. This study demonstrates that alcohols lower the temperature required for the maximal activation of such a promoter and that the concentration of alcohol required decreases as its hydrophobicity increases. A similar correlation has been found between the members of this alcohol series and their effect on a range of membrane functions. Our results therefore indicate that perturbation of the cell membrane may play a role in the heat induced activation of this HSE-containing promoter.
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Synthesis of yeast cell wall glucan and evidence for glucan metabolism in a Saccharomyces cerevisiae whole cell system
More LessThe synthesis and metabolism of yeast cell wall glucan were studied using a Saccharomyces cerevisiae construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with in vitro findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected. In contrast, cycloheximide, a known ps inhibitor, displayed an elevated ratio. This whole cell construct affords a simplified system for elucidating the synthesis and metabolic activity of the yeast cell wall and enables the discrimination between specific effectors of gs and ps.
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Enzyme inactivation related to a hyperoxidant state during conidiation of Neurospora crassa
More LessThe conidiation process of Neurospora crassa is characterized by three morphogenetic steps: hyphal adhesion, aerial hyphal formation, and production of conidia. Previous data indicated the occurrence of a hyperoxidant state at the onset of all three morphogenetic steps. Because glutamine synthetase (GS) and the biosynthetic glutamate dehydrogenase [GDH(NADP)] enzymes are susceptible to inactivation by reactive oxygen species, we followed these enzyme activities during conidiation and under different physiological conditions and related them to the hyperoxidant states and morphogenesis. Loss of GS activity occurred prior to all three morphogenetic steps, coinciding with an increase in total protein oxidation. Oxidized GS polypeptides were detected during hyphal adhesion. Loss of GDH(NADP) activity also occurred during hyphal adhesion and before aerial hyphal formation; the enzyme polypeptide and activity decreased in the adhered hyphae to low values and no GDH(NADP) was detected in aerial hyphae. The catabolic GDH [GDH(NAD)] behaved in an opposite manner, increasing its activity during hyphal adhesion and aerial hyphae development. These results are discussed with regard to cell differentiation and the conidiation process in N. crassa.
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Adaptation of Aspergillus niger to several antifungal agents
Adaptation of Aspergillus niger to short-term stress induced by three antifungal agents [amphotericin B (AMPH), miconazole (MCZ), and ketoconazole (KCZ)] was observed and evaluated quantitatively using individual hyphae. Spores were inoculated onto a poly-l-lysine-coated glass plate making up the base of a culture vessel. Potato dextrose broth (PDB) was added and the vessel incubated for 24 h at 28 °C. The growth rate of an arbitrarily selected test hypha was measured automatically. Exposure to AMPH (0.075 μg ml-1) stopped the growth of the hypha. After washing with PDB, the same concentration of AMPH was applied again. The growth of the test hypha was not inhibited. This phenomenon was defined as adaptation to the short-term stress of AMPH. Similarly, adaptation was observed with MCZ (0.01 μg ml-1) and KCZ (0.5 μg ml-1). The time required for the test hypha to restart growth after washing with PDB depended upon the concentration of MCZ or KCZ, but not upon the concentration of AMPH.
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Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and ProP
More LessExogenously supplied L-pipecolic acid was accumulated by Escherichia coli cells and protected them while growing at inhibitory osmolarity. Using specific uptake mutants and competitive assays, we established that the imino acid enters the cells through the ProP and ProU systems with K m values of 225 and 53 μM, respectively. Surprisingly, in spite of the requirement for the wild-type proX gene for osmoprotective ability, no binding activity of labelled pipecolate with the periplasmic protein encoded by proX could be detected. In an attempt to demonstrate whether the two porters (ProP and ProU) are the only carriers involved in osmoregulation, a variety of molecules known for their intracellular osmolarity-dependent accumulation in various organisms were investigated. N-Dimethylproline (proline betaine), N-dimethylglycine, homobetaine (β-alanine betaine), γ-butyrobetaine and dimethylsulfonio-propionate were found to be capable of promoting the growth of osmotically stressed E. coli. All of these molecules enter bacterial cells via ProP and ProU porters. None of the osmoprotectants except N-dimethylproline was able to bind the periplasmic protein encoded by proX, while this protein was necessary for their uptake. Apparently, ProP and ProU are the sole osmoporters involved in osmolyte influx into E. coli cells.
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Low-temperature-induced adaptations in fatty acid metabolism of Acanthamoeba castellanii cultures of different ages: relationship to changes in cell division, oxygen uptake and phagocytotic activity
More LessThe degree of unsaturation of membrane lipids of Acanthamoeba castellanii at 30 °C decreased with batch-culture age. These changes were primarily attributable to a decline in microsomal Δ12-desaturase activity and in the relative proportion of linoleate. No change in the fatty acid composition, Δ12-desaturase activity or any increased incorporation of [1-14C]acetate into polyunsaturated fatty acids was observed following chilling of early- and mid-exponential-phase cultures to 15 °C. In contrast, chilling of late-exponential and stationary-phase cultures resulted in a rapid and marked increase in the synthesis of polyunsaturated fatty acids. Thus, after 12 h incubation at 15 °C, the relative proportions of oleate and linoleate in these older cultures were similar to those of early- and mid-exponential-phase cultures at 30 °C. Despite these differences, an approximately 9.5 h lag in cell division was evident following chilling of both mid-exponential and late-exponential/early-stationary-phase cultures, and the subsequent pattern of cell division over 60 h incubation was similar in both cases. Oxygen uptake rates in cultures of either age were also decreased approximately equally at 15 °C. In contrast, chilling of mid-exponential-phase cells resulted in only an approximately 44% reduction in the rate of phagocytosis of fluorescently-labelled latex beads, whereas an approximately 98% inhibition of phagocytosis by late-exponential/early-stationary-phase cells resulted at 15 °C. Furthermore, a gradual subsequent increase in the rate of phagocytosis at 15 °C was only observed in the older cultures and was correlated with increases in the fatty acid unsaturation index of these cells. Thus, 12 h after chilling, the cells from late-exponential/early-stationary-phase cultures had achieved rates of phagocytosis similar to those of chilled cells from mid-exponential growth and their fatty acid compositions were now similar. The results suggest a role of membrane lipid unsaturation in the control of phagocytotic activity of A. castellanii.
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Transport of mannose by an inducible phosphoenolpyruvate: fructose phosphotransferase system in Streptococcus salivarius
More LessStreptococcus salivarius transports mannose by a phosphoenolpyruvate: sugar phosphotransferase system (PTS) which consists of a membrane Enzyme II and two forms of Enzyme III (IIIMan) with molecular masses of 38.9 kDa (IIIMan H) and 35.2 kDa (IIIMan L) respectively. Using a pseudorevertant (strain 57P) isolated from a IIIMan L-deficient spontaneous mutant unable to grow on mannose, we demonstrated that S. salivarius could also transport mannose by an inducible fructose PTS. This PTS phosphorylated fructose at the C-1 position with a high affinity (10 μM) and mannose at the C-6 position with a low affinity (200 μM). Derepression of this system in some IIIMan L-deficient mutants would explain their ability to grow on mannose.
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Design and analysis of chemostat experiments using Metabolic Control Analysis: a top-down approach
More LessThis paper is concerned with the application of Metabolic Control Analysis to chemostat experiments. It deals with the peculiar problems which are presented when using a chemostat for such purposes. The paper shows how to apply a top-down approach to quantifying control in the chemostat exerted by both the ‘physical’ components (e.g. dilution rate) and the biological components. The analysis is considered at a number of levels, depending on the amount of information on the components of metabolism either known or accessible experimentally. A method is also presented for using the information obtained by this analysis to obtain a ‘virtual’ control coefficient, which is defined as a control coefficient which would be obtained if it were possible to clamp all metabolites ‘external’ to the micro-organism, including biomass, at their steady-state levels. This allows, amongst other things, control coefficients with respect to growth rate to be determined under chemostat conditions.
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The physiological function of periplasmic glucose oxidation in phosphate-limited chemostat cultures of Klebsiella pneumoniae NCTC 418
More LessPeriplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen-and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) activities of cell-free extracts of glucose-grown cells harvested from aerobic chemostat cultures limited in various nutrients showed that at least a tenfold overcapacity in GAPDH activity was present under phosphate-limited conditions with respect to the steady-state carbon fluxes through this enzyme. The physiological significance of this adaptation and the possible role of GDH and GaDH are discussed.
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Carbohydrate metabolism and physiology of the parasitic protist Trichomonas vaginalis studied in chemostats
More LessThe parasitic protist Trichomonas vaginalis was cultured in chemostats with glucose or maltose as carbon and energy source. The maximum growth rate was about six divisions per day independent of the substrate, and the apparent K m for glucose was 0.375 mM. While growing on maltose, the growth rate depended linearly on the maltose concentration, indicating that in contrast to glucose metabolism a diffusion step is rate-limiting to maltose metabolism. Cultures were examined over a wide range of growth rates under four conditions: utilizing glucose or maltose as carbon and energy source, with the carbon source rate-limiting or present in excess. Cell density, cellular protein and carbohydrate content as well as residual substrate concentration in the culture fluid were measured at each steady state. The protein content was constant at 100 μg protein per cell except when T. vaginalis was cultured under glucose limitation; in the latter case, slow-growing cells had less protein than cells grown at high rates. When growing under glucose limitation T. vaginalis metabolism changed to become more energy efficient at growth rates exceeding about half the maximum rate. The maintenance energy at the low growth rates accounted for approximately half of the total carbon consumption, which is high in comparison to other micro-organisms. At low growth rates the yield on maltose exceeded that on glucose, when expressed in terms of carbon equivalents. The yields on maltose and glucose were equal, but much lower, when the carbon source was not rate-limiting. A comparison of the data of this study with similar studies on other organisms suggests that the high maintenance energy of T. vaginalis may be used primarily for maintaining homeostasis of the internal conditions to enable growth and survival in the vagina.
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Adaptation of the carbon metabolism of Trichomonas vaginalis to the nature and availability of the carbon source
More LessThe anaerobic parasitic protist Trichomonas vaginalis was adapted in chemostats to eight different conditions defined by different growth rates and carbon regimes. Glucose or maltose was used as carbon and energy source. Cells cultured under well-defined steady states were tested in short-term experiments. The kinetics of glucose and maltose uptake were determined and their glucokinase and α-glucosidase activities were measured. Uptake in 20 min was measured with radiolabeled glucose and maltose, rather than analogues, using the silicone oil centrifugation technique. Hence, the accumulated label represents both transport and metabolic activity. The total uptake of glucose was highest in organisms that had been starved for glucose during growth. The kinetics of glucose uptake can be understood by assuming rate-limitation by transport across the plasma membrane at low external concentrations and by the subsequent metabolism at concentrations exceeding a cross-over value. The specific glucokinase activity correlated in only four out of eight cases with the saturation uptake. The kinetics of maltose uptake indicated rate-limitation at low maltose concentrations by a diffusion-limited step and at higher levels by metabolic steps. The uptake of maltose was primarily affected by the growth rate during culture, the highest growth rates resulting in most uptake. Maltose uptake was determined only partially by the cellular α-glucosidase activity. The activities of both transport and metabolic enzymes changed due to the culture conditions suggesting that the control over glucose and maltose metabolism is shared by several steps in the pathway.
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- Plant-Microbe Interactions
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The Rhizobium leguminosarum biovar viciae nodO gene can enable a nodE mutant of Rhizobium leguminosarum biovar trifolii to nodulate vetch
More LessAnalysis of the nodulation characteristics of transposon-induced mutants of Rhizobium leguminosarum bv. viciae revealed that nodO and the closely-linked rhi genes contribute to nodulation of peas (Pisum sativum) and the vetch Vicia hirsuta. Although mutation of nodO alone had no significant effect on nodulation of either legume, a double mutant lacking both nodO and nodE nodulated both legumes very poorly. Similarly, a double mutant lacking nodE and either rhiA or rhiB nodulated peas less efficiently than a nodE mutant. Thus, although mutations affecting only the rhi genes normally have no observed effect on nodulation, these genes do appear to contribute to pea nodulation. When transferred to a wild-type strain of Rhizobium leguminosarum bv. trifolii, neither nodO nor the rhi gene region conferred pea or vetch nodulating ability. However, in a nodE mutant of R. I. bv. trifolii, nodO did confer a significant level of vetch nodulating ability, indicating that the secreted NodO protein can play a role in determining legume recognition by R. I. bv. viciae.
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- Genome Analysis
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Determination of genome size and a preliminary physical map of an extreme alkaliphile, Micrococcus sp. Y-1, by pulsed-field gel electrophoresis
More LessLarge restriction fragments of genomic DNA from Micrococcus sp. Y-1 were separated by pulsed-field gel electrophoresis (PFGE). Since Micrococcus sp. Y-1 has a G + C content of approximately 70%, restriction fragments were obtained by digesting chromosomal DNA with endonucleases which recognize A+T-rich sequences. Five enzymes, Sspl, Spel, Xbal, Hpal and EcoRI, were used for generation of distinctly separated fragments in the size range 100-500 kb. No site for DraI was detected. In contrast, sites for 8-base-recognizing enzymes, but not for NotI and Sfil, were frequent. The genome size of Micrococcus sp. Y-1 was determined from restriction fragments separated by PFGE, and was estimated to be approximately 4061 kb. Partial digestion experiments revealed the order of the six Sspl fragments on the chromosome.
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Cloning and nucleotide sequencing of a 15 kb region of the Bacillus subtilis genome containing the iol operon
More LessWithin the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced. This region (14974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase. All the genes identified are transcribed in the same direction as that of the movement of the replication fork. A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B. subtilis), and PhoB and FtsE proteins (Escherichia coli). It also implied that two genes (B65B and B65E) might encode a set of two-component regulatory proteins and that the B65F gene might encode a protein belonging to the ATP-binding cassette (ABC) family. Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted.
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Two identical copies of IS1246, a 1275 base pair sequence related to other bacterial insertion sequences, enclose the xyl genes on TOL plasmid pWWO
More LessTwo identical direct repeats of a 1275 bp sequence, designated IS1246, encompass the xyl genes, which determine the catabolism of toluene, m- and p-xylenes to central metabolites, on the TOL catabolic plasmid pWWO. IS1246 has a terminal inverted repeat of 12 bp (5‘GGGCACCTCGAA3’) and contains a major open reading frame of 280 codons. This ORF shows significant homology with ORFs encoded by a number of bacterial insertion sequences from Bacteroides, Neisseria and Escherichia coli.
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