- Volume 140, Issue 10, 1994
Volume 140, Issue 10, 1994
- Review Article
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- Microbiology Comment
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- Biochemistry
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Chemical characterization of lipopolysaccharides from Legionella feeleii, Legionella hackeliaeand Legionella jordanis
More LessLipopolysaccharides (LPS) from Legionella feeleiiserogroup 1, L. hackeliaeserogroup 1 and L. jordaniswere subjected to chemical analysis. All three LPS contained D-mannose, D-glucose, D-glucosamine, L-glycero-D-manno-heptose, 2-keto-3-deoxyoctonic acid and glycerol. In addition the LPS of L. feeleiiwas characterized by L-quinovose (tentatively identified) and L-fucosamine, L. hackeliaeLPS by D-quinovosamine, D-galactosamine and D-galacturonic acid, and L. jordanisLPS by D-quinovosamine. Phosphorylated sugars were detected in all three LPS. The backbone sugar of the lipid A part was in each case 2,3-diamino-2,3-dideoxy-D-glucose substituted with a complex pattern of fatty acid, including 20-22 different amide-linked (non-branched and methyl-branched) 3-hydroxy fatty acids of chain-length ranging from 12 to 23 carbon atoms. The fatty acid patterns included also ester-linked nonhydroxylated entities and the uncommon 27-oxo-octacosanoic acid and 29-oxotriacontanoic acid. The LPS of L. hackeliaeand L. jordanisalso contained heptacosane-1,27-dioic and nonacosane-1,29-dioic acid, and their 2-hydroxy analogues were characteristic of L. jordanisLPS. SDS-PAGE patterns of the three LPS were distinctly different. Both L. feeleiiand L. jordanisproduced smooth-form LPS with characteristic ladder patterns, whereas L. hackeliaeLPS were of more rough-type character.
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Fatty acid biosynthesis in novel ufamutants of Neurospora crassa
New mutants of Neurosporacrassa having the ufaphenotype have been isolated. Two of these mutants, like previously identified ufamutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novosynthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufamutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.
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Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora
More LessThe white-rot basidiomycete Ceriporiopsis subvermisporaproduces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pl values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pl 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II). mainly the MnPs with higher pl values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermisporawhen growing on wood chips of Pinus radiata.Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pl values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pl values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pl values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF. Immunoblotting studies showed that the molecular mass of MnP isoenzymes from liquid cultures was about 52.5 kDa, whereas the molecular masses of MnPs extracted from wood varied from 52.5 kDa to 62.5 kDa upon ageing of the cultures. The amino terminal sequences of seven MnP isoenzymes were determined. The consensus sequences of MnPs from liquid and solid cultures were clearly distinct, although both showed homology to MnPs from related white-rot fungi.
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Evidence that fimbriae of the smut fungus Microbotryum violaceumcontain RNA
More LessThe cells of the fungus Microbotryum violaceumproduce many long, fine surface hairs that are similar in size and morphology to bacterial pili or fimbriae. These fungal fimbriae are assembled from 74 kDa glycoprotein subunits. We now present evidence that these fimbriae also have a RNA component. Isopycnic centrifugation of fimbriae in caesium chloride produced one band at a density intermediate to that of protein and nucleic acid. The absorbance spectrum of the intact fimbriae was consistent with that of a nucleoprotein. After extraneous RNAs were enzymically removed from the purified fimbrial preparation, disruption of the fibrils resulted in the release of not only the 74 kDa glycoprotein subunits, but also a 30 base single-stranded RNA species. To our knowledge, this is the first example of extracellular RNA as a component of a surface appendage.
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The nematophagous fungus Verticillium chlamydosporiumproduces a chymoelastase-like protease which hydrolyses host nematode proteins in situ
More LessThe nematophagous fungus Verticillium chlamydosporiumsecreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M r33000, pl 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a K mfor Suc-(Ala)2-Pro-Phe-pNA of 4.3 × 10-5M and a k catof 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situfrom the outer layer of the egg shell of the host nematode Meloidogyne incognitaand exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporiumand V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium.These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.
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Chemical modification of Bacillus thuringiensisactivated δ-endotoxin and its effect on toxicity and binding to Manduca sextamidgut membranes
More LessThe effect of chemical modification of aromatic and basic residues of the Bacillus thuringiensisinsecticidal CrylA(c) toxin on toxicity and receptor binding was studied. Modification of four or more of the 43 arginine residues resulted in abolition of toxicity towards Manduca sextaand Pieris brassicaein vivoand a Choristoneura fumiferanacell line in vitro.Modification of seven or more of the 27 tyrosine residues resulted in reduction of toxicity. Upon modification of most of the 10 tryptophan residues, toxicity was reduced. Modification of histidine residues had no effect on toxicity. A quantitative binding assay was developed and optimized to compare the binding of derivatives with native toxin to M. sextabrush border membrane vesicles. The reduction or abolition of toxicity observed upon selective modification of tyrosine or arginine residues was reflected in the binding abilities of the derivatives. However a non-toxic derivative, modified at tryptophan residues, retained its ability to bind vesicles. These results suggest that tyrosine and arginine residues are involved in the binding interaction of toxin with receptor but tryptophan residues are involved in some subsequent step.
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Prokaryotic triterpenoids: new hopanoids from the nitrogen-fixing bacteria Azotobacter vinelandii, Beijerinckia indicaand Beijerinckia mobilis
More LessThree nitrogen-fixing bacteria, Azotobacter vinelandii, Beijerinckia indicaand Beijerinckia mobilis, were shown to contain large amounts of triterpenoids of the hopane series. In A. vinelandii, the major compound was a novel bacteriohopanepentol ether accompanied by a similar bacteriohopanetetrol derivative: in both compounds, the hopanoids are linked via an ether bond to a carbapseudopentose moiety often found in bacterial hopanoids. In the two Beijerinckiaspecies, diplopterol and 2β-methyldiplopterol were accumulated in much larger amounts than those usually recorded in hopanoid-producing eubacteria, while 2β-methyldiploptene was isolated for the first time from B. mobilis.Whereas in B. mobilisaminobacteriohopanetriol was the only C35hopanoid, the simultaneous presence of bacteriohopanetetrol and aminobacteriohopanetriol in B. indicais a rather unusual feature.
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Luteolin uptake by Rhizobium meliloti: evidence for several steps including an active extrusion process
More LessEvidence for several steps in luteolin uptake by Rhizobium melilotiis presented. The first step is highly reversible and strongly pH dependent absorption, greatly enhanced at acidic pH. Studies with liposomes suggest that this step consists of a passive dissolution of the flavonoid in its non-ionized state in the lipidic matrix of the membrane. This first step is enhanced in bacteria killed by high temperature. The second step, a slower and more stable accumulation, is not pH dependent. The nature of the sites involved in this more stable binding remains to be determined. Low temperature and treatment with the metabolic inhibitor potassium cyanide resulted in an increase of luteolin uptake and a decrease of extrusion of flavonoids, suggesting an active extrusion process. Studies with spheroplasts or isolated membranes showed that under physiological conditions most of the luteolin was entrapped in the outer membrane while only a small amount was kept in the cytoplasmic membrane. Arguments are presented supporting the view that R. melilotihas developed a mechanism to keep luteolin in its membranes which helps avoid the toxic effect of the flavonoid on the respiratory chain by trapping most of it in the outer membrane and to maintain the nodoperon in a transcriptionally active form in conditions where this strong inducer is no longer exuded by the host plant.
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- Environmental Microbiology
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In situprobing of Gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides
More Less23S-rRNA-targeted oligonucleotide probes were designed for the phylogenetic group ‘Gram-positive bacteria with high G + C content of DNA’ (GPBHGC). A sequence idiosyncrasy in two adjacent base pairs in the stem of helix 69 in domain IV of the 23S rRNA is present in all hitherto analysed strains of GPBHGC. An oligonucleotide probe targeted to this region hybridized only with strains of GPBHGC and was successfully used for in situmonitoring of these cells in activated sludge. Another unique feature of the 23S rRNA molecules of GPBHGC is a large insertion in domain III. Fluorescent oligonucleotides targeted to the highly variable regions of the rRNA within the insertions of Corynebacterium glutamicumDSM 20300T, Aureobacterium testaceumDSM 20166 and Brevibacteriumsp. DSM 20165 hybridized specifically to their target strains, whereas probing with oligonucleotides complementary to the rRNA-coding strand of the 23S rDNA and to the spacer between 16S and 23S rRNA of C. glutamicumdid not result in detectable fluorescence. This confirmed that the large 23S insertions are indeed present in 23S rRNAs of GPBHGC and provide potential target sites for highly specific nucleic acid probes.
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- Genetics And Molecular Biology
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Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli
More LessAn acnAmutant of Escherichia coliwas constructed by replacing the chromosomal acnAgene by an internally deleted derivative containing a kan Rcassette. Southern and Western blotting confirmed that the acnAgene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed. However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconitase activity. This activity was due to an analogous unstable enzyme(s) designated aconitase B. Studies on the regulation of aconitase A synthesis using an acnA-lacZtranslational fusion showed that the acnAgene resembles other citric acid cycle genes in being subject to CRP-mediated catabolite repression and ArcA-mediated anaerobic repression. In addition to being activated by the SoxRS oxidative stress regulatory system, the acnAgene appeared to be activated by the ferric uptake regulator (Fur). It was concluded that the acnAgene belongs to at least four global regulatory networks, crp, arcA, furand soxRS. In contrast, the aconitase B activity decreased after exposure to oxidative stress and was less affected by anaerobiosis. Comparable studies with the fumarase genes (fumA, Band C) indicated that fumA(encoding the unstable aerobic iron-sulphur-containing fumarase) is activatedby the ferric uptake regulator (Fur) and fumC(encoding the stable fumarase) is activated by the SoxRS oxidative stress regulatory system.
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Isolation of the Thiobacillus ferrooxidans ntrBCgenes using a T. ferrooxidans nifH-lacZfusion
More LessAn agar plating technique was developed in which the activation of expressior of a Thiobacillus ferrooxidans nifH-lacZgene fusion was used to isolate the ntrBCgenes from a T. ferrooxidansgene library. An Escherichia coli ntrCmutant containing the nifH-lacZfusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T. ferrooxidans ntrBCgenes. A 4.47 kb region from the T. ferrooxidanschromosome was sequenced. Analysis of the sequence revealed that the ntrBand ntrCgenes were closely linked to a third ORF of unknown function. Analysis of the 900 bp region upstream of the T. ferrooxidans ntrBCgenes and Southern hybridization experiments confirmed that in T. ferrooxidansATCC 33020, the glnAand ntrBCgenes are unlinked. Expression of the T. ferrooxidans nifH-lacZfusion in E. coliwas activated in the presence of the T. ferrooxidans ntrBCgenes and regulated by nitrogen.
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Transcriptional regulation of the four promoters of the agarase gene (dagA) of Streptomyces coelicolorA3 (2)
More LessThe agarase gene (dagA) of Streptomyces coelicolorA3(2) is transcribed from four promoters that are recognized by at least three, and probably four, different RNA polymerase holoenyzmes, each containing a different factor. S1nuclease protection studies revealed that transcription from all four promoters is induced by the products of agar hydrolysis and strongly repressed by glucose. Mutants deficient in glucose kinase activity were defective in glucose repression of all four promoters. Mutants were isolated or identified in which transcription from all four promoters had become inducer-independent (i.e. constitutive), establishing the existence of a repressor gene for dagAthat does not appear to be located within 9 kb of the structural gene. The cloned dagAgene was also constitutively expressed in the closely related strain Streptomyces lividans, which does not normally make agarase and which presumably lacks the repressor gene. Glucose was still able to repress dagAtranscription even under conditions of constitutive expression, suggesting that glucose kinase does not mediate its effect via inducer exclusion. Relative differences in the use of the four promoters were not detected during different stages of growth of surface-grown cultures, although dagAtranscription appeared to peak during the production of aerial mycelium.
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Possible function and some properties of the CcpA protein of Bacillus subtilis
More LessThe ccpAmutations alsA1(alsA1is allelic to ccpA) and ccpA::Tn917completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis. The alsA1mutation also partially affected catabolite repression of sporulation. Analysis of revertants from the alsA1mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide −14 to adenine within the Shine-Dalgarno sequence of the ccpAgene (ccpAtranslation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying the ccpAgene was cloned into Escherichia coliplasmid pUC19 and B. subtilisplasmid pUB110, resulting it plasmids pCCPA19 and pCCPA110, respectively. The ccpAgene carried in pCCPA110 complemented the alsA1mutation. Western blotting revealed that the level of the CcpA protein in B. subtiliscells, which seemed to be constitutively synthesized, was approximately 10 times lower for the alsA1mutant than for the wild-type. The CcpA protein synthesized by either E. colicells bearing pCCPA19 or B. subtiliscells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose The molecular mass of the protein purified from E. coliwas 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cissequence for catabolite repression of the gntoperon, but it bound non-specifically to DNA.
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An investigation of plasmids from Staphylococcus aureusthat mediate resistance to mupirocin and tetracycline
More LessPlasmids conferring mupirocin resistance were prepared from isolates of Staphylococcus aureusobtained from four patients in the same ward. The plasmids are related and in all of them the gene conferring mupirocin resistance (mupA) is flanked by copies of IS257in direct repeat. In two plasmids mupA and IS257have been duplicated and in one of these plasmids (pJ3358) a small pT181-like plasmid conferring tetracycline resistance is present flanked by copies of IS257.Filter mating with a strain containing pJ3358 as donor and selection on tetracycline sometimes resulted in transfer of the pT181-like plasmid containing a copy of IS257.Analysis showed that the pT181-like plasmid with the insertion of IS257is present in high copy number and that the IS257element is inserted in the copy number control region of the plasmid.
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Characterization of ø393-A2, a bacteriophage that infects Lactobacillus casei
More LessBacteriophage ø393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. ϕ393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3′-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole ϕ393-A2 genome has been cloned in Escherichiacoli using plasmid- and phage-derived cloning vectors.
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Cloning and nucleotide sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckiisubsp. lactisDSM 7290, and partial characterization of the enzyme
More LessCell extracts of Lactobacillus delbrueckiisubsp. lactisDSM 7290 were found to exhibit unique peptolytic ability against unusual β-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli.Recombinant clones harbouring pepVwere identified by their ability to allow the utilization of carnosine (β-alanyl-histidine) as a source of histidine by the E. colimutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV.A 2.4 kb fragment containing pepVwas subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepVor its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepVis of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coliand Saccharomyces cerevisiae.
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A Lactococcus lactisgene encodes a membrane protein with putative ATPase activity that is homologous to the essential Escherichia coli ftsHgene product
More LessA gene, encoding a protein homologous to an essential Escherichia coliprotein, FtsH, was identified adjacent to the hptgene and the trnAoperon in the Gram-positive bacterium Lactococcus lactis.The deduced amino acid sequence of the gene product showed full-length similarity to FtsH of E. coli, Yme1p of Saccharomycescerevisiae and a conserved region found in a new family of putative ATPases. In-frame fusions of L. lactis ftsHand phoA1in E. coli, and immunodetection of the L. lactisFtsH protein in cell fractions using anti-E. coliFtsH serum showed that L. lactis ftsHwas expressed and encodes a membrane protein. When contained on a high copy number plasmid, the L. lactis ftsHgene complemented the lethality of a δftsH3::kanmutation in E. coliat 37 °C and below, indicating that the L. lactis ftsHgene can functionally replace the E. coli ftsHgene to some extent. The resulting E. colistrain showed temperature sensitivity and salt sensitivity. A L. lactismutant with an insertion into ftsHwas salt-, heat- and cold-sensitive. These results suggest that FtsH is somehow involved in stress responses. Southern hybridization analysis indicated that genes homologous to ftsHof L. lactiswere also present in Bacillus subtilis, and several Lactobacillusand Leuconostocspecies, suggesting high conservation of ftsHin bacterial species.
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Regulation of the gene encoding translation elongation factor 3 during growth and morphogenesis in Candida albicans
The level of the TEF3mRNA, which encodes the fungal-specific translation elongation factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans.In contrast to a previous report, TEF3mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25 °C to 37 °C) combined with the addition of 10% (v/v) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25 °C to 37 °C) combined with an increase in the pH of the medium (from pH 4.5 to 6.5). TEF3mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast to cultures undergoing morphological changes. TEF3mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in TEF3expression that accompany the dimorphic transition in C. albicansappear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se.For this reason TEF3mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation factor might be subject to post-transcriptional regulation.
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Glucose-transport-deficient mutants of Schizosaccharomyces pombe: phenotype, genetics and use for genetic complementation
More LessGlucose-transport-deficient mutants of Schizosaccharomycespombe were obtained by treatment of wild-type cells (972h-) with N-methyl-Ns'-nitro-N-nitrosoguanidine, and by selection of resulting mutants on gluconate medium containing 0.05% 2-deoxy-D-glucose (2DG). One mutant, designated YGS-B22, was unable to grow on D-glucose and/or D-fructose as a carbon source (Glc/Fru-), and was resistant to 2DG; hence, none of the three sugars was taken up by the mutant cells. The hexokinase activity in the wild-type and the mutant cells was equal. Genetic purification of YGS-B22by back-crossing with a leucine-auxotrophic mutant and the wild-type resulted in two strains: YGS-4, with reduced 2DG resistance, and YGS-5, which had lost 2DG-resistance. YGS-5grew in D-glucose-containing media, albeit very slowly. No measurable sugar uptake was detectable in either of the two mutants within the 1 h test interval. Tetrad analyses proved a Mendelian segregation of growth on D-glucose and leucine auxotrophy. However, 2DG resistance did not co-segregate with the Glc/Fru-phenotype, indicating that the transport deficiency and 2DG resistance characters are not encoded on the same genomic locus. Using a genomic bank of Sch. pombe, two transformants, YGS-5-G7and YGS-5-G12, were found which had regained the wild-type growth and transport phenotype by complementation. Correspondingly, both D-glucose uptake and 2DG accumulation were restored in the transformed strains. Restriction analysis and Northern blots suggested that the G7 genomic fragment and the 4.1 kb SalI restriction fragment of the G12 genomic fragment both contain a complete structural symporter gene.
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Arabinase gene expression in Aspergillus niger: indications for coordinated regulation
More LessAspergillus nigersecretes three glycosylated glycosyl hydrolases which are involved in degradation of the plant cell wall polysaccharide L-arabinan: α-L-arabinofuranosidases (ABF) A and B, and endo-1,5-α-L-arabinase (ABN) A. The nucleotide sequence of the previously cloned gene encoding ABF A (abfA) from A. nigerwas determined. The coding region contains seven introns. Mature ABF A comprises 603 amino acids with a molecular mass of 65.4 kDa as deduced from the nucleotide sequence. The secreted enzyme is N-glycosylated. The primary structures of the three A. nigerarabinases characterized lack similarity. Regulation of arabinase expression upon induction by sugar beet pulp and by L-arabitol was studied as a function of time. This was done in wild-type A. nigeras well as in transformants carrying multiple copies of either one of the ABF-encoding genes. Each arabinase gene responded differently upon a mycelial transfer to L-arabitol-containing medium. Extra copies of abfAor abfBled to a decreased expression level of ABN A, though the repression elicited by abfBis stronger and more persistent than that effected by abfA.Multiple copies of both abfgenes influence expression of the other ABF similarly, but to a far less pronounced degree than they affect ABN A synthesis. Four putative promoter elements, shared by all three arabinase genes, could be involved in coordination of L-arabinan degradation by A. niger.
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Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis
More LessThe parasitic protozoon Trichomonas vaginalisproduces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from T. vaginalisgenomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a T. vaginaliscDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5′ end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5′ ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the T. vaginalisgene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of Dictyostelium discoideum, human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains α and β). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to D. discoideumCP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy. Similarly sized, single abundant transcripts were present for all four sequences. Overall, the data show that we have identified a family of genes in T. vaginaliswhich encode a number of CPs. In total, seven distinct sequences have been recognized. This suggests that the multiplicity of CP activities seen
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Insertion sequence IS1137, a new IS3 family element from Mycobacterium smegmatis
A new insertion sequence (IS) has been isolated from Mycobacterium smegmatis.It is 1361 bp long and possesses characteristics of the IS3 family elements. It harbours 32 bp imperfect inverted repeats at its extremities and a 3 bp direct repeat flanks the element, possibly as the result of a transposition event. This IS, IS1137, contains three major ORFs. Two of them, ORF A and ORF B show homologies both at the amino acid sequence level and at the organization level with the ORFs encoding the transposase of the IS3 family elements. IS1137 has a narrow host range and was found only in M. smegmatisand M. chitae.The fact that IS1137 is not present in the M. tuberculosiscomplex strains makes this element a new candidate for transposon mutagenesis in mycobacteria.
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The ribosomal RNA (rrn) operons of fast-growing mycobacteria: primary and secondary structures and their relation to rrnoperons of pathogenic slow-growers
More LessThe two ribosomal RNA (rrn) operons (rrnAand rrnB) of Mycobacterium smegmatiswere investigated. The leader regions, part of the 16S rRNA genes, the spacer-1 regions, part of the 23S rRNA genes, and the spacer-2 regions were amplified by PCR or by inverse PCR and the products were cloned and sequenced. No differences in the sequences of the two operons were detected downstream from the Box A antitermination element of the leader region. Upstream from Box A a slow-grower-like Box B antitermination element was found in rrnAbut not in rrnB.Primer extension experiments revealed that the start of transcription lies at least 370 nucleotides upstream from the 5′-end of the 16S rRNA gene and an RNase processing site near to the Box A element. Secondary structures were deduced for pre-16S rRNA and pre-23S rRNA which are distinct from, but closely related to, the corresponding structures of slow-growing mycobacteria. On the basis of these results it is proposed that the emergence of the slow-growers from the main mycobacterial line was coincident with the deletion of a segment of DNA spanning an rrnB-like operon, leaving an rrnA-like operon as the sole source of rRNA. An explanation is also proposed for the need for two Box A motifs in the transcription of an rrnoperon based on competition between the polymerase and the nascent 30S subunit for either protein S10 and/or Box A sequences.
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- Pathogenicity And Medical Microbiology
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Adherence of Ureaplasma urealyticumto human epithelial cells
More LessAdherence of Ureaplasma urealyticumcells to eukaryotic cell monolayers was quantified using the Bertholet assay to monitor ammonia produced from urea by ureaplasma urease. Adherence was abolished by pre-treatment of ureaplasmas with HeLa cell extracts and inhibited to varying degrees by pre-treatment of the ureaplasmas with N-acetylneuraminic acid, specific antisera and monoclonal antibodies. The data suggest the presence of several ureaplasma adhesins, some of which are species-or serotype-specific and some of which are proteinaceous and antigenic. The serotype-8-specific 96 kDa surface-expressed antigen may be one adhesin. Pre-treatment of HeLa cell monolayers with neuraminidase significantly reduced ureaplasma adherence and, using a novel ‘immunoblot adherence assay’, ureaplasmas were shown to bind to a number of HeLa cell components, three of which appear to terminate in sialic acid.
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The susceptibility of S-layer-positive and S-layer-negative Aeromonasstrains to complement-mediated lysis
More LessForty strains of Aeromonas hydrophilaand Aeromonas veroniirecovered from invasive and non-invasive infections were tested for their susceptibility to complement-mediated lysis by 65% pooled human serum (PHS). Based upon the results of this assay, two major populations could be defined. The first group (n= 20) consisted of serogroup 0:11 strains, all of which possessed a paracrystalline surface layer (S layer); all of these strains were refractory to the bactericidal activity of 65% PHS with the exception of A. hydrophilastrain AH-121, which was composed of mixed subpopulations of serum-susceptible and serum-resistant clones. A second collection of isolates (n= 20), all of which were S-layer-negative, contained a subgroup of strains (n= 7) that were highly susceptible to complement-mediated lysis, showing a greater than 100-fold reduction of viable progeny within 30 min of exposure to 65% PHS. Serum-resistant strains from both groups could not be lysed by exposure of bacterial cells to polyclonal somatic or whole cell antisera or to 30 μg ml-1of polymyxin B nonapeptide prior to challenge with 65% PHS. Analysis of selected serum-resistant and serum-susceptible strains from both groups showed that all isolates activated the complement pathway and most bound C3b to the cell surface, indicating that the inability of complement to lyse serum-resistant strains was related to a defect in the terminal portions of the complement pathway. The major differences noticed between serum-resistant and serum-susceptible strains were a lack of a definable lipopolysaccharide side chain profile and higher 50% lethal dose values in strains that were susceptible to complement-mediated lysis.
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- Physiology And Growth
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Concomitant appearance of intrinsic thermotolerance and storage of trehalose in Saccharomyces cerevisiaeduring early respiratory phase of batch-culture is CIF1-dependent
More LessStrains of Saccharomyces cerevisiaethat exhibit varied capacities for accumulation of trehalose were tested for intrinsic thermotolerance. Yeast that accumulated trehalose rapidly in early respiratory phase showed equally rapid attainment of thermotolerance, whereas a strain unable to accumulate trehalose at this stage of culture showed markedly delayed appearance of thermotolerance. These results were obtained using closely related but non-isogenic diploids and so it is possible that variable factors other than trehalose were responsible for the observed thermotolerance effects. Therefore, a pair of isogenic diploid S. cerevisiaestrains was generated to facilitate further testing of whether trehalose functions in intrinsic stress tolerance. Both isogenic strains inherited a partially reverted cif1phenotype, designated CPR, from the trehalose-deficient progenitor that had been used in construction of the non-isogenic strains. The CPR phenotype permitted growth on glucose but not accumulation of trehalose, indicating that not all cif1-related deficiencies were suppressed in the CPR strains. However, one of the isogenic CPR pair was cif1/cif1and failed to accumulate trehalose, whilst the other was cif1/CIF1and was able to accumulate this sugar. The trehalose-proficient strain showed intrinsic stress tolerance whereas the trehalose-deficient strain was sensitive to heat stress during early respiratory growth. These results suggest that one or more functions of CIF1, not operating in the cif1/cif1(CPR) strains, are important for intrinsic thermotolerance of yeast in early respiratory phase. When considering these results with those of others whose work has indicated a role for trehalose in protection of proteins and membranes, it is reasonable to hypothesize that the trehalose deficiency associated with cif1/cif1(CPR) strains could be a key factor in their intrinsic thermosensitivity. However, if this is the case the importance of trehalose, relative to other stress tolerance factors, appears to vary with growth phase and culture status.
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Nitrite causes reversible inactivation of nitrate reductase in the yeast Hansenula anomala
The addition of nitrite, the product of the reaction catalysed by nitrate reductase, to cell suspensions of the yeast Hansenula anomalacaused a reversible inactivation of NADPH-dependent nitrate reductase activity. The haem- and Mo-dependent and Mo-dependent activities of nitrate reductase, determined with the non-physiological electron donors FMNH2and reduced methyl viologen respectively, were less affected. A similar inactivation was found with the proton ionophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The inactive enzyme was found in the particulate fraction and cosedimented with the mitochondrial fraction. When the NADPH-dependent nitrate reductase activity was restored in vivothe enzyme was found in the soluble fraction. The inactivation of nitrate reductase by nitrite, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone was dependent on the external pH. The treatment of isolated mitochondria at alkaline pH with Triton X-100 solubilized about 30% of the inactive enzyme.
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Vesicles prepared from Streptococcus mutansdemonstrate the presence of a second glucose transport system
More LessStreptococcus mutans, an important aetiological agent of dental caries, is known to transport glucose via the phosphoenolpyruvate (PEP) phosphotransferase system (PTS). An alternative non-PTS glucose transport system in S. mutansIngbritt was suggested by the increased ATP-dependent phosphorylation of glucose and the presence of higher cellular concentration of free glucose in cells grown in continuous culture under PTS-repressed conditions compared to those resulting in optimal PTS activity. A method was developed for the preparation of membrane vesicles in order to study this system in the absence of PTS activity. These vesicles had very low activity of the cytoplasmic enzymes, glucokinase, pyruvate kinase and lactate dehydrogenase. This, coupled with the lack of glycolytic activity and the inability to transport glucose, suggested that the vesicles would also be deficient in PTS activity because of the absence of the general soluble PTS proteins, Enzyme I and HPr, required for the transport of all PTS sugars. Freeze fracture electron microscopy and membrane H+-ATPase analysis indicated that over 90% of the vesicles had a right-side-out orientation. Vesicles from cells grown in continuous culture under PTS-dominant and PTS-repressed condition both exhibited glucose counterflow. This indicates the presence of a constitutive non-PTS carrier in the organism capable of transporting glucose and utilizing ATP for glucose phosphorylation. Analysis of growth yields of cells grown under PTS-repressed and PTS-optimal conditions suggests that ATP or an equivalent high energy molecule, must be involved in the actual transport process. This analysis is consistent with an ATP-binding protein model such as the Msmtransport system reported by R. R. B. Russell and coworkers (J Biol Chem267, 4631-4637), but it does not exclude the possibilit of a separate permease for glucose.
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Characteristics of Helicobacter pylorigrowth in a defined medium and determination of its amino acid requirements
More LessA defined medium has been developed for Helicobacter pylorithat gives growth characteristics (growth rate, maximum cell number and maximum colony-forming-unit count) comparable to those in a complex medium (Isosensitest broth+5%, v/v, foetal bovine serum). Differences found in the death rate reflected a partial (50%) conversion to a coccoid cell form of the organism in the stationary and death phase in the defined medium, versus the almost complete (> 99%) conversion seen in the complex medium. The medium was used to study the amino acids required for growth by 10 strains of H. pylori.All strains required arginine, histidine, isoleucine, leucine, methionine, phenylalanine and valine, and eight of the strains also required alanine; five of the strains required serine. In the absence of glucose none of the 20 amino acids tested elicited growth when added at high concentration. However, in the presence of glucose, alanine induced considerably enhanced growth over that seen in the control, consistent with its use either as a nitrogen source or possibly an additional carbon source. The medium described will facilitate investigations into the metabolism and physiology of H. pylori, previously only possible with sophisticated approaches such as nuclear magnetic resonance spectroscopy.
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The utilization of thiocyanate as a nitrogen source by a heterotrophic bacterium: the degradative pathway involves formation of ammonia and tetrathionate
More LessA Gram-negative soil bacterium (isolate 26B) has been shown to utilize up to 100 mM thiocyanate as a source of nitrogen when supplied with glucose as the source of carbon and energy. During growth of isolate 26B with thiocyanate as the source of nitrogen, no ammonia, nitrate, nitrite, cyanide, cyanate, sulfate, sulfite, sulfide or carbonyl sulfide was detected in the growth medium. Growth of the bacterium on 14C-labelled thiocyanate (1.6 μCi) and glucose, yielded 14C-labelled carbon dioxide (0.9 μCi). The addition of 2.9 mM thiocyanate to a bacterial suspension in phosphate buffer (50 mM, pH 7.4) resulted in the utilization of 2.1 mM thiocyanate and the production of 2.0 mM ammonia. This activity was inducible and only occurred after growth of the bacterium with thiocyanate as the source of nitrogen. Tetrathionate (0.7 mM) was detected in the medium after the utilization of thiocyanate (2.4 mM) by a suspension of the bacterium in phosphate buffer, and thiosulfate (1.0 mM) was detected as an intermediate. The addition of sulfide or thiosulfate to the bacterial suspension also resulted in the formation of tetrathionate. The utilization of both of these compounds appeared to be constitutive. A pathway for thiocyanate utilization by isolate 26B is proposed which involves the hydrolysis of thiocyanate to produce cyanate and sulfide. The cyanate then undergoes further hydrolysis to form ammonia and carbon dioxide. The sulfide is ultimately oxidized to tetrathionate via a pathway which includes thiosulfate.
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Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi
More LessThe entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliaeand Paecilomyces farinosuswere cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon I-1for 30 d at 25 °C. The water activity (aw) of the media ranged from 0.925 to 0.998. Growth of M. anisopliaeand P. farinosuswas stimulated between 0.975 and 0.995 awon glucose media and that of P. farinosusat 0.975 awon glycerol media. At < 0.970 aw’growth of each fungal species was significantly reduced (P< 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1for B. bassiana, 77.3 and 90.3 mg g-1for M. anisopliae, and 26.7 and 76.1 mg g-1for P. farinosus.The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon I-1than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1on glucose media with 142.7 g added carbon I-1, depending on species. Conversely, conidia of B. bassianaand P. farinosuscontained maximum amounts of trehalose (≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon I-1and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon I-1, conidia of B. bassianacontained up to 154.0 mg glycerol plus erythritol g-1. When B. bassianaand P. farinosuswere grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliaecontained < 17.0 mg trehalose g-1under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.
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Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum
More LessMutants of Rhizobium leguminosarumbv. viciaeMNF300 and R. leguminosarumbv. trifoliiWU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb EcoRI-PstIDNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase (pobA) from Pseudomonasspp. and Acinetobacter.Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic ‘drag’ mechanism dependent on the activity of the pobAgene product.
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Iron acquisition during growth in an iron-deficient medium by Rhizobiumsp. isolated from Cicer arietinum
More LessA catechol-type siderophore was produced extracellularly by Rhizobiumsp. strain BICC 651 isolated from Cicer arietinumduring growth in iron-deficient medium. Production of the siderophore was fully repressed in the presence of 50 μM Fe3+. The siderophore was purified and characterized as containing 2,3-dihydroxybenzoic acid (DHBA) as the core compound and threonine as its conjugate. The siderophore was able to reverse growth inhibition of the strain induced by ethylenediamine-di(o-hydroxyphenyl-acetic acid) (EDDA). A high-affinity iron-transport system capable of transporting 59Fe-siderophore complex was also induced in RhizobiumBICC 651 grown under iron deficiency. Two protein bands of molecular masses 76 and 82 kDa were also inductively synthesized in the outer membrane of the cells. A partially purified ferrireductase enzyme of RhizobiumBICC 651 catalysed reductive release of iron from the ferric chelate of DHBA. The enzyme had a K mof 0.3 mM for ferri-DHBA, was constitutive in nature, and was present in the cytosolic fraction during growth under both iron-deficient and iron-sufficient conditions. The enzyme occurred as two isoenzymes with R Fvalues of 0.48 and 0.51, respectively, in a nondenaturing polyacrylamide gel.
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- Plant-Microbe Interactions
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Characterization of a 14 kDa component with low expression in a unique Nod+ Fix- Bradyrhizobium japonicum
More LessA pronase-sensitive, 14 kDa component of bacteroids of Bradyrhizobium japonicumI-110ARS was identified and characterized using monoclonal antibodies. This component was weakly synthesized or was missing in bacteroids of a unique Nod- Fix- mutant, ARS2525. Both the 14 kDa component and poly-β-hydroxybutyrate (PHB) were located in the same protein peak after sucrose density gradient separation of lysed bacteroids. Thirty-five-day-old bacteroids of B. japonicumI-110ARS contained up to 10 times more PHB than B. japonicumARS2525 bacteroids. Immunocytochemistry of ultra-thin nodule sections showed that the component was associated with PHB granules in the bacteroids, possibly with the limiting membrane around the granules. This observation implies that the component may be similar to other storage lipid body proteins. Whether low synthesis of the 14 kDa component or low PHB content was a cause, a consequence or related to the Fix- phenotype was not established.
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myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarumbv. viciae
More LessOn the basis of enzyme assays, myo-inositol appears to be catabolized via 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in Rhizobium leguminosarumbv. viciae, as occurs in Klebsiellaaerogenes. The first two enzymes of the pathway, myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase were increased 7- and 77-fold, respectively, after growth of R. leguminosarumon myo-inositol compared to glucose. Cells of R. leguminosarumgrown on glucose as the carbon source and then resuspended in myo-inositol, increased myo-inositol-dependent O2consumption by sixfold in 3 h, confirming this to be an inducible pathway. Succinate, malate and glucose exhibited strong catabolite repression of the myo-inositol catabolic pathway with myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase being repressed by at least 68% and 80%, respectively, in all cases. A dicarboxylate transport mutant (dctA) was unable to repress either enzyme when grown on myo-inositol and succinate. There was no repression of the first two enzymes of the myo-inositol catabolic pathway in the background of constitutive expression of the dicarboxylate transport system, indicating a dicarboxylate must be present to cause repression. The data imply that dicarboxylates are required intra-cellularly to repress these enzymes. Three transposon mutants were isolated that cannot grow on myo-inositol as the sole carbon source and were unable to induce either myo-inositol dehydrogenase or 2-keto-myo-inositol dehydratase. The mutants were unaffected in the ability to nodulate peas and vetch. Furthermore, vetch plants infected with one mutant (RU360) reduced acetylene at the same rate as plants infected with the wild type. Bacteroids did not oxidize myo-inositol, nor were the catabolic enzymes detected, confirming myo-inositol is not important as an energy source in bacteroids. The possible role of myo-inositol in the rhizosphere is considered.
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Identification of chromosomal genes located downstream of dctDthat affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum
More LessIn Rhizobium leguminosarumboth the C4-dicarboxylate transport system and wild-type lipopolysaccharide layer (LPS) are essential for nitrogen fixation. A Tn5 mutant (RU301) of R. leguminosarumbv. viciae3841, was isolated that is only able to synthesize LPS II, which lacks the O-antigen. Strain RU301 exhibits a rough colony morphology, flocculates in culture and is unable to swarm in TY agar. It also fails to grow on organic acids, sugars or TY unless the concentration of calcium or magnesium is elevated above that normally required for growth. The defects in the LPS and growth in strain RU301 were complemented by a series of cosmids from a strain 3841 cosmid library (pRU3020-pRU3022) and a cosmid from R. leguminosarumbv. phaseoli8002 (pIJ1848). The transposon insertion in strain RU301 was shown to be located in a 3 kb EcoRI fragment by Southern blotting and cloning from the chromosome. Sub-cloning of pIJ1848 demonstrated that the gene disrupted by the transposon in strain RU301 is located on a 2.4 kb EcoRI-Pstlfragment (pRU74). R. leguminosarumbv. viciaeVF39-C86, which is one of four LPS mutants previously isolated by U. B. Priefer (1989, J Bacteriol171, 6161-6168), was also complemented by sub-clones of pIJ1848 but not by pRU74, suggesting the mutation is in a gene adjacent to that disrupted in strain RU301. Complementation and Southern analysis indicate that the region contained in pIJ1848 does not correspond to any other cloned lpsgenes. Two dctAmutants, RU436 and RU437, were also complemented by pIJ1848 and pRU3020. Mapping of pIJ1848 and Southern blotting of plasmid-deleted strains of R. leguminosarumrevealed that dctDand the region mutated in strain RU301 are located approximately 10 kb apart on the chromosome. Analysis of homogenotes demonstrated that there is not a large region important in calcium utilization, organic acid metabolism or LPS biosynthesis located between the gene disrupted in strain RU301 and dctD.Strain VF39C-86 also required an elevated concentration of calcium for growth on succinate, while strains mutated in the α-chromosomal or β-plasmid group of lpsgenes grew at the same calcium concentrations as the wild type, demonstrating that the additional calcium requirement is not a property of all LPS rough mutants. Strain RU301 nodulates peas, but does not reduce acetylene, demonstrating that the gene mutated in this strain is essential for nitrogen fixation.
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- Systematics
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Bacterial hopanoids from pink-pigmented facultative methylotrophs (PPFMs) and from green plant surfaces
More LessSix strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) isolated from phylloplane surfaces of different plants were analysed for the presence of triterpenoids of the hopane series. All of the cultures produced hopanoids in abundant quantities and contained the same compounds as the type strain of Methylobacterium organophilum: diplopterol, 2β-methyldiplopterol, bacteriohopanetetrol, a tetrol glycoside and two tetrol ethers. The presence of a guanidinium group on the carbapseudopentose moiety of one of these ethers and/or of 2β-methyldiplopterol seems to be restricted to the genus Methylobacterium.Small amounts of bacteriohopanepolyols were detected in three of seven plants studied. Since no bacterial C35hopanoids have been reported in eukaryotes, we believe they are probably derived from eubacterial epibionts present on the phylloplane surfaces, the most numerous of which are Methylobacteriumspp.
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Permeabilization of mycolic-acid-containing actinomycetes for in situhybridization with fluorescently labelled oligonucleotide probes
More LessThe application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrousand Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coliand Pseudomonas putidawere also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37°C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coliand P. putidawere rendered permeable in only 10 min. Interestingly, L. plantarumcould not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.
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A numerical phenotypic taxonomic study of the genus Neisseria
More LessA numerical phenotypic taxonomic study of 315 strains of Neisseriaand some allied bacteria examined for 155 phenotypic tests showed 31 groups, most of which were reasonably distinct. These fell into four major areas. Areas A, B and C contained species of Neisseria, whereas area D contained the organisms known as ‘false neisserias’ together with Branhamella, Moraxellaand Kingellaspecies. Area A contained N. gonorrhoeae(which showed two subgroups), N. meningitidis(with two subgroups, and N. cinereaclosely associated), N. polysaccharea, N. elongatasubsp. glycolyticaand N. lactamica.Area B contained mainly organisms from the human nasopharynx, and the nine groups were not very distinct: only three, N. mucosa, N. perflavaand N. siccacould be recognized by the presence of type strains, and there was little relationship between taxonomic position and species epithets. Area C contained several groups from animals, N. animalis, N. canisand two phenons that may be justified as new species of Neisseria, one from lizards and the other from dental plaque of herbivores. Area C also contained N. elongata, N. subflava(with N. flavescens), type strain of Morococcus cerebrosisand the CDC groups M-5 (N. weaveri) and EF-4. Area D contained Branhamella catarrhalis, a combined group which consists of strains of the ‘false neisserias’N. caviaeand N. cuniculi, the ‘false neisserias’ N. ovis, and a group of Moraxellastrains. A small group representing Kingella kingaeis included in area D. Mean test error was 1.7%.
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- Genome Analysis
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Pulsed-field gel electrophoresis analysis of the genome of amino acid producing corynebacteria: chromosome sizes and diversity of restriction patterns
More LessA large number of species of corynebacteria are known to be amino acid producers, including members of the genera Corynebacteriumand Brevibacterium.Pulsed-field gel electrophoresis (PFGE) of DNA fragments obtained by using endonucleases which recognize AT-rich hexanucleotide or octanucleotide sequences produces a discrete pattern of bands useful for fingerprinting and physical mapping of the chromosome. Using Pacland Swalendonucleases the genome of Brevibacterium lactofermentumATCC 13869 (genome size 3052 kb) was consistently cut into 26 and 20 bands, respectively, and the genome of Corynebacterium glutamicumATCC 13032 (2987 kb) yielded 27 and 26 fragments, respectively. The pattern of restriction fragments was identical for related strains (B. lactofermentumATCC 13869, B. lactofermentumBLO, B. lactofermentumR31) but different from the pattern of fragments of other soil isolates of the same species (B. lactofermentumDSM 20412) or from closely related organisms such as C. glutamicum; the different pattern of restriction fragments may be used to differentiate taxonomically related species. Brevibacterium linensshowed a different behaviour, due to its high G + C content; its genome (3105 kb) was resolved into 8 or 15 fragments, respectively, by digestion with the hexanucleotide-recognizing endonucleases Draland Asel.PFGE of DNA fragments obtained using these enzymes is a powerful technique for quick resolution of the corynebacteria genome into a small number of large fragments.
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)