- Volume 139, Issue 8, 1993
Volume 139, Issue 8, 1993
- Review Article
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- Biochemistry
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Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes
More LessSUMMARY: A dissimilatory sulphite reductase was isolated from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus. In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an α2β2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide. The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm. We have cloned and sequenced the genes for the α and β subunits of this enzyme, which we designate dsrA and dsrB, respectively. They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea. dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene. Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-[Fe4S4] complexes in sulphite reductases and nitrite reductases from other species. The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the α subunit binds a sirohaem-[Fe4S4] complex, and chemical analyses showed the presence of only two sirohaems per α2β2 enzyme molecule. Both deduced peptides also contain an arrangement of cysteine residues characteristic of [Fe4S4] ferredoxins, and chemical analyses were consistent with the presence of six [Fe4S4] clusters per α2β2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.
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Membrane-associated NADH dehydrogenase activities in Rhodobacter capsulatus: purification of a dihydrolipoyl dehydrogenase
More LessSUMMARY: The presence of several NADH dehydrogenase activities associated with cytoplasmic membrane vesicles of chemoheterotrophically grown Rhodobacter capsulatus MT1131 was demonstrated by combining isoelectric focusing with NADH-tetranitrobluetetrazolium activity staining, a procedure that should have general applicability in the analysis of bacterial NADH dehydrogenase activities. Low pI (pI = 5.7), Mid pI (pI = 6.9) and High pI (pI = 8.5) bands were resolved. The Mid pI NADH dehydrogenase activity was purified and identified as a dihydrolipoyl dehydrogenase. Our data indicate that this dihydrolipoyl dehydrogenase is derived from a 2-oxoacid dehydrogenase complex which is associated with the cytoplasmic membrane.
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Molybdenum uptake in Escherichia coli K12
More LessSUMMARY: Molybdenum uptake was examined in Escherichia coli K12 using the radionuclide 99Mo. The molybdenum uptake system was characterized in an unusual chlD strain, which appeared to be normal in uptake of the MoO%2−% 4 ion but altered in subsequent molybdenum processing. As a consequence, molybdenum could be chased from cells in the chlD strain, while it was irreversibly assimilated in the wild-type strain. Molybdenum uptake showed a biphasic kinetic curve, with a very rapid binding followed by a slow uptake phase. The uptake appeared to involve an active transport system. Molybdenum, probably in the form of molybdate, accumulated by a factor of about 30 in the cells. An energy source was necessary and uptake was inhibited by arsenate, but not by CCCP (carbonyl cyanide m-chlorophenylhydrazone). The uptake system saturated with a Km of 2·5·2·7 × 10−8 M. Uptake seemed to depend on a periplasmic binding protein, since cold shock treatment and arsenate abolished uptake. A molybdate binding protein activity was detected in the periplasmic fluid with a KD of 9 nM. Sulphate inhibited uptake and the uptake activity was pH dependent, with an apparent pK of 6·7. These results imply that molybdate transport belongs to the family of energy-dependent periplasmic binding protein systems. An explanation for the peculiar behaviour of the chlD strain used in this work is proposed.
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- Biotechnology
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Membrane lipid composition and invertase secretion of Neurospova crassa and its wall-less mutant slime: effects of temperature and the surfactant Tween 80
More LessSUMMARY: The effects of temperature and the surfactant Tween 80 on the secretion of invertase by the ascomycete fungus Newospora crassa and its wall-less strain slime were investigated. Temperature acclimation dramatically affects the phospholipid fatty acid pattern in both strains. The levels of polyunsaturated fatty acids in membrane lipids of wild-type Neurospova crassa and slime increased as growth temperature decreased. Chromatogram analysis from cultures acclimated to 15 C showed high levels of linolenic acid (18:3), and low levels of oleic acid (18:1), suggesting desaturation. Reducing the temperature during growth to 15 C affected phospholipid fatty acid composition in both strains, which resulted in a higher level of invertase secretion. The wild-type Newospora crassa showed no difference in invertase secretion in the presence of Tween 80. However, the addition of the surfactant to slime cultures caused a 60 % increase in invertase secretion, which was more evident after 48 h incubation.
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- Environmental Microbiology
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Attachment of a Pseudomonas-like bacterium and Bacillus coagulans to solid surfaces and adsorption of their S-layer proteins
More LessSUMMARY: The role of S-layer proteins in bacterial adhesion to solid surfaces was investigated by determining whether there was a relationship between the adsorption of S-layer protein and the attachment of the source bacteria to a series of substrata exhibiting a range of water-wettabilities. Polystyrene substrata, prepared by treatment with H2SO4, provided advancing water contact angles ranging from 76 to 46. The test bacteria were a Pseudomonas-like strain, designated EU2, and the thermophilic bacterium Bacillus coagulans. In two out of four cases, S-layer adsorption paralleled cell attachment. Numbers of attached EU2 and amount of S-layer adsorption in phosphate buffer both increased with increasing substratum hydrophobicity. Numbers of attached B. coagulans and S-layer adsorption in distilled deionized water both decreased with increasing substratum hydrophobicity. The inconsistencies in attachment and S-layer adsorption observed in the remaining experiments were possibly due to the fact that S-layer proteins were free to adsorb by both inner and outer faces, whereas S-layer on the cell could adsorb only by the outer face. The results indicated that S-layers may play a role in bacterial adhesion to surfaces, but that the adhesiveness of S-layers depends upon their specific chemistry and environmental conditions such as medium composition and temperature.
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Adhesion to nematodes of conidia from the nematophagous fungus Drechmeria coniospora
More LessSUMMARY: Conidia of the endoparasitic nematophagous fungus Drechmeria coniospora adhere to the sensory organs of many nematode species. In some cases the adhesion phase is followed by penetration of the nematode cuticle and subsequent infection. In a study of eight different nematode species and five strains of the fungus only two species were infected: Panagrellus redivivus was infected by all strains and Ditylenchus dipsaci was infected by four strains, although the conidia of all fungal strains adhered to all of the nematode species tested. Treatment of the nematode P. redivivus and the conidia of D. coniospora with proteases gave a decreased adhesion in contrast to glycosidases, lipases and other enzymes tested. Inhibitory effects on adhesion were obtained after treatment of conidia with the carbohydrate N-acetylneuraminic acid; and the amino acids alanine and proline. Hydrophobicity and electrical charge appear not to be involved in conidial adhesion. A previous hypothesis on the presence of a sialic-acid-specific lectin in this interaction appears to be incorrect and the present results indicate no involvement of carbohydrates in the adhesion process. The results suggest that the adhesion is mediated by protein(s) in the adhesive part of the conidium binding to protein(s) excreted from the sensory organs of the nematodes.
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- Genetics And Molecular Biology
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Detection of seven species of pathogenic leptospires by PCR using two sets of primers
SUMMARY: Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemoirhagiae-derived primers (G1/G2) enabled amplification of DNA from L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-II) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and G2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-II, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.
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Molecular analysis of a flagellar core protein gene of Serpulina (Treponema) hyodysenteriae
SUMMARY: The flaB2 gene encoding a protein located in the core of the periplasmic flagella of Serpulina hyodysenteriae was cloned and sequenced. The FlaB2 protein consists of 285 amino acids and has a calculated molecular mass of 31.1 kDa. Southern blot analysis indicated that at least one, and possibly two genes related to flaB2 are present in the genome of S. hyodysenteriae. Comparison of the amino acid sequence of FIaB2 to sequences present in data banks showed significant similarity with the core flagellins of other spirochaetes, in particular with a FIaB2 protein from Treponema phagedenis.
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The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters
More LessSUMMARY: Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria. Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression. In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes. In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA. Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA. Sequences flanking the K4 capsule genes were found in the chromosome of all E. coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic.
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The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae
More LessSUMMARY: The complete nucleotide sequence of pSR1, a 3 kb multicopy cryptic plasmid from Corynebacterium glutamicum ATCC 19223 has been determined. pSR1 is unrelated to the 4.4 kb Brevibacterium lactofermentum plasmid pBL1 and shows no DNA sequence conservation with plasmids from Staphylococcus. Transposon insertion and deletion mutants located the minimal replicon to within a 2.1 kb NcoI-BclI restriction fragment. This region contains a single large open reading frame, ORF2, flanked at the 5 end by a series of inverted repeat sequences which may modulate its expression, and at the 3’ end by a region which may contain a replication origin. ORF2 (position 1633-2636) with a maximum coding potential of 36 kDa is essential for pSR1 replication and was designated the rep gene. The predicted ORF2 protein product exhibits 47% identity over a length of 343 amino acids with a replication-associated ORF in the C. diphtheriae plasmid pNG2, many of the changes being in the third base position. This observation suggests that pSR1 and pNG2, which are two plasmids from environmentally separated Corynebacterium species, may share a common ancestral rep gene.
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The ancestral IncP replication system consisted of contiguous oriV and trfA segments as deduced from a comparison of the nucleotide sequences of diverse IncP plasmids
More LessSUMMARY: In most plasmids which have been studied to date the functions required for plasmid replication are clustered in a 2—3 kb region. However, in all known naturally occurring plasmids of the Escherichia coli incompatibility group P the essential replication functions, oriV, the vegetative replication origin and trfA, which encodes proteins essential to activate oriV, are separated by blocks of DNA consisting of either known genes conferring resistance to antimicrobial agents and/or putative transposable elements. Nucleotide sequence comparisons reported here reveal that these blocks of DNA have inserted at different points into a backbone of DNA common to IncP plasmids. The results indicate that in the common ancestor of present IncP plasmids oriV and trfA must have been contiguous, whilst a ρ-independent transcriptional terminator, now lost in IncPα plasmids, may have prevented trfA operon transcription from interfering with the activity of oriV.
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Characterization of an IS-like element from Mycobacterium tuberculosis
More LessSUMMARY: A DNA sequence, present in members of the Mycobacterium tuberculosis complex, has been identified and characterized. The distribution of this DNA sequence among mycobacterial species was analysed by DNA hybridization and PCR experiments. As the sequence was detected only in bacteria belonging to the M. tuberculosis complex, it may be useful for the rapid discrimination of mycobacteria. Interestingly, the sequence has some characteristics of an insertion element (IS) and codes for a hypothetical protein with significant homologies to proteins encoded by several IS elements of other organisms, namely IS427 and IS869 from Agrobacterium tumefaciens, IS402 from Pseudomonas cepacia, Tn4811 from Streptomyces lividans and ISRm4 from Rhizobium meliloti. Together, these elements form a previously unrecognized family of transposable elements. This finding suggests the possibility of horizontal gene transfer between pathogenic mycobacteria and other organisms including Gram-negative plant-pathogenic bacteria.
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Genetic and biochemical characterization of the two glutamine synthetases GSI and GSII of the phosphinothricyl-alanyl-alanine producer, Stveptomyces viridochromogenes T494
More LessSUMMARY: The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.
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Cloning, nucleotide sequence and expression in Stveptomyces lividans and Escherichia coli of pabB from Lactococcus lactis subsp. lactis NCDO 496
More LessSUMMARY: A gene (pabB) encoding the aminase activity of p-aminobenzoate (PABA) synthase in Lactococcus lactis subsp. lactis was cloned in pIJ41 and expressed in Streptomyces lividans strains defective in PABA biosynthesis. Expression of the gene was associated with a 1.2 kb deletion between the aph promoter and the cloning site in pIJ41. Subcloning in pBR322 and expression in Escherichia coli AB3295 of the cloned L. lactis DNA fragment localized the pabB-complementing gene in a 1.9 kb segment. The nucleotide sequence of this segment contained a 1410 bp open reading frame encoding a 470-amino-acicl polypeptide of 50937 Da. The deduced amino acid sequence showed substantial similarity to those reported for PabB and TrpE from several organisms. Synonymous codon usage reflected the low G + C content in the genomic DNA of L. lactis subsp. lactis, and therefore differed markedly from the preferred usage in the S. lividans host. The cloned heterologous pabB DNA was expressed in amounts that allowed accumulation of excreted PABA in cultures of S. lividans transformants.
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Mutants of Escherichia coli affected in respiration: the cloning and nucleotide sequence of ubiA, encoding the membrane-bound p-hydroxybenzoate: octaprenyltransferase
More LessSUMMARY: A mutant of Escherichia coli has been isolated that is unable to grow aerobically on non-fermentable substrates, but able to grow anaerobically on glycerol with alternative electron acceptors such as fumarate. Nitrate as electron acceptor supports anaerobic growth on glycerol, but not on succinate or lactate. Oxygen consumption rates by cell-free extracts with succinate, lactate or glycerol 3-phosphate as substrates were low relative to activities in an isogenic control strain but were restored in vitro by adding ubiquinone-1. Transformation of the mutant with a cloned 2.6 kb ClaI-PvuII fragment of chromosomal DNA restored cellular quinone levels and growth on succinate. The plasmid also complemented a previously isolated ubiA mutant for aerobic growth on non-fermentable substrates. The nucleotide sequence of the cloned fragment revealed a fragment of plsB (91.7 min on the E. coli chromosome map) and three open reading frames (ORFs), one of which (ORF3) encodes a protein with a predicted molecular mass of 32511 Da. The hydrophobicity profile of the ORF3 protein is characteristic of a membrane protein with five hydrophobic regions and is very similar to that of the Saccharomyces cerevisiae COQ2 gene product (p-hydroxybenzoate:polyprenyltransferase, required for the second step of ubiquinone biosynthesis) and to the product of the E. coli cyoE gene. Complementation of ubi mutants with various deletion derivatives of the cloned DNA fragment confirms that ORF3 is ubiA. ORF3 is closely linked to ubiC (ORF2), which encodes chorismate lyase.
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Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene
More LessSUMMARY: The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66). This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide. The enzyme was purified from F. lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE). mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated. This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible. Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone. A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E. coli using the expression vector pGEX-2T. Features of F. lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented.
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Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production
More LessSUMMARY: Coronatine-producing and non-producing strains of Pseudomonas syringae pv. glycinea have been examined. We found a connection between copper resistance and synthesis of coronatine. Published data implied that these properties may be encoded on different plasmids. Production of coronatine and copper resistance were also found to be correlated for pv. glycinea in 19 field-isolates from leaf spots of plants in a soybean field and in 28 strains of a bacterial culture collection. Genomic diversity within pv. glycinea was investigated by plasmid profiling, DNA hybridization studies and PCR analysis. All strains unable to produce coronatine (cor-) were sensitive to copper ions and showed no homology to DNA from plasmid pSAY1, which carries a gene cluster for steps in coronatine production. In addition, cor- strains could be distinguished from coronatine-producing strains by a single unique band when amplified by random primer PCR. Plasmid profiles of strains isolated from field-populations during 1983, 1985 and 1990 showed that coronatine-producing and non-producing strains were present. The plasmid patterns also varied in 28 strains examined from a culture collection. No correlation between plasmid patterns and race specificity was observed. Cosmid pSAY1 proved to be an effective probe for detection of the coronatine synthesis genes and also revealed polymorphisms in coronatine producing strains of pv. glycinea.
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- Pathogenicity And Medical Microbiology
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Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms
More LessSUMMARY: Chromosomal DNA from 37 Leptospiracaea representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, Clal and EcoRI. The Southern blots were hybridized with a biotinylated E. coli 1·5 kb 16S rDNA probe and gave 36 reproducible and unique patterns. With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no. 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, Clal and EcoRI restriction fragment length polymorphisms (RFLP). L. interrogans and L. borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L. parva incertae sedis were distinct and separate. Strains representing L. biflexa sensu lato presented divergent RFLP patterns. A porcine isolate was identified to be L. interrogans pomona Pomona.
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Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of Legionella pneumophila
More LessSUMMARY: A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using L. pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L. pneumophila-specific antigens on the surface of E. coli. Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was L. pneumophila DNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of the E. coli parent, the L. pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L. pneumophila but not in E. coli JM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using L. pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L. pneumophila polypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designated ompM. Furthermore, using the fertile chicken egg virulence assay, clone LP 116 producing the 25 kDa MOMP of L. pneumophila showed an increase in virulence when compared to the E. coli parent strain.
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Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes
J. Frey, J. T. Bosse, Y.-F. Chang, J. M. Cullen, B. Fenwick, G. F. Gerlach, D. Gygi, F. Haesebrouck, T. J. Inzana, R. Jansen, E. M. Kamp, J. Macdonald, J. I. MacInnes, K. R. Mittal, J. Nicolet, A. N. Rycroft, R. P. A. M. Segers, M. A. Smits, E. Stenbaek, D. K. Struck, J. F. van den Bosch, P. J. Willson and R. YoungSUMMARY: The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for Actinobacillus pleuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9,10 and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxI operon will have the designations apxIC, apxIA, apxIB, and apxID for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII. ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular mass between 103 and 105 kDa. The genes of the apxII operon will have the designations apxIIC for the activator gene and apxIIA for the structural toxin gene. In the apxII operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxIB and apxID of the apxI operon. The designation ApxIII is proposed for the non-haemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin III (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat). This protein is strongly cytotoxic and has an apparent molecular mass of 120 kDa. The genes of the apxIII operon have the designations apxIIIC, apxIIIA, apxIIIB and apxIIID for the activator gene, the structural gene and the two secretion genes respectively.
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Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide
More LessSUMMARY: The class 1 outer-membrane protein of Neisseria menigitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype PI. 16b epitope have been synthesized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.
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Migration of rat peritoneal cells after intra-abdominal infection with Bacteroids fragilis and Escherichia coli
More LessSUMMARY: A fibrin clot model for intra-abdominal abscess formation was used to study the migratory properties of peritoneal cells from rats during the early stages of infection. Peritoneal cells and fibrin clot remnant were harvested 6 h after implantation of a sterile, singly infected (Escherichia coli or Bacteroides fragilis) or mixed infected (E. coli and B. fragilis) fibrin clot. Histological study of fibrin clots, removed 6 h after implantation, showed a deeper infiltration by host cells of B. fragilis infected clots compared to the others. This difference in infiltration by peritoneal cells was not due to differences in fibrinolytic activity of the bacterial strains. Differential cell counts of the peritoneal cells from rats implanted with sterile, singly and mixed infected fibrin clots showed distribution over subpopulations to be independent of the bacterial content of the infected clots used. In vitro migration assays showed no significant differences in migration by peritoneal cells from rats implanted with clots containing a different bacterial composition. Since B. fragilis infected fibrin clots were more deeply infiltrated by host defence cells than the other clots, and only mixed infected clots led to persistent abscesses in this model, we conclude that local conditions within the fibrin matrix rather than intrinsic cellular capacities of the host cells are important for the process of abscess formation.
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Interaction of Propionibacterium acnes with skin lipids in vitro
More LessSUMMARY: Propionibacterium acnes is the predominant microbial resident within the pilosebaceous follicles of sebum-rich areas of human skin. This study investigated the effects of known hydrophobic components of sebum on the physiology and nutrition of this microorganism, grown anaerobically at 33 °C, under defined conditions using continuous culture techniques. The medium used was chemically defined, comprising eight amino acids, with glucose as the main carbon energy source, and the culture pH was maintained at 5.6. The range of sebum lipids assayed was based on the C18 monounsaturated fatty acid 9-cis-octadecenoic acid (oleic acid). Stock micronized solutions were aseptically pulsed into continuous cultures in the presence and absence of glucose, and nutritional effects monitored. None of the lipid substrates significantly affected P. acnes growth either in terms of maximum specific growth rate (μmax) or final culture biomass yield. Glycerol (3 mg ml-1) was found to be a poor carbon/energy source in comparison to glucose. Bacterial cells did, however, adhere with varying degrees, to the different lipid species, with maximum adherence occurring with the free fatty acid. This observation was confirmed by preliminary uptake experiments using [14C]oleic acid. The interactive site for cell adherence may be the lipid-fibrillar layer associated with the cell surface of P. acnes, as discerned in electron microscopical studies. The findings of this investigation suggest that one function of the P. acnes lipase may be to aid colonization within the pilosebaceous follicle, by promoting cell adherence to components such as oleic acid.
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- Physiology And Growth
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Zonation in migrating magnetococci
More LessSUMMARY: Zone formation and movement of cells from mixed, natural colonies of magnetococci were studied. We concluded that (i) zoning resulted from hydrodynamic interaction between cells swimming in parallel as a result of magnetotaxis and (ii) magnetic interaction between cells had a role in determining the form and behaviour of zones. Our evidence is as follows. The formation of zones normal to the direction of movement during magnetotaxis requires that a component of the magnetic field be directed into a surface, resulting in cells being concentrated at the surface, facilitating cell interaction. The orientation of the surface with respect to gravity is irrelevant, excluding gravitation as a critical factor. Quasi-stable lateral associations between pairs of cells with similar speeds were observed, confirming a prediction that hydrodynamic interaction between cells could bring about such associations, and have a major role in the production of more extensive lateral associations including zones. Cells travelling much faster than a zone were observed to pass through it, although with some delay, suggesting that cells with speeds near that of the zone would be captured. In populations of slowly moving magnetococci, head-to-tail associations between cells were observed, indicating that at low swimming speeds magnetic instead of hydrodynamic interaction was dominant. When the direction of the magnetic field was reversed, the direction of zone movement was also reversed, and zones became narrower and more sharply defined for a few seconds. This was interpreted as a consequence of a magnetic effect of the mass of cells in a zone on cells at the zone edge. We also observed instability and gravitational fall of a dense layer of cells overlying cell-free fluid (Rayleigh-Taylor instability). This is well known with eukaryotic micro-organisms but has not been previously reported for bacteria.
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Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12
A. Darwin, P. Tormay, L. Page, L. Griffiths and J. ColeSUMMARY: The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified. No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity. The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain. A double mutant defective in both FdhN and FdhH retained less than 10 % of the activity of the FdhN+FdhH+ strain. No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain. It is concluded that all three of the known formate dehydrogenases of E. coli can contribute to the transfer of electrons from formate to the Nrf pathway. Mutants defective in Nrf activity and cytochrome c 552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E. coli Genetic Stock Center. The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity. Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF. Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity. From these clones, plasmids were isolated that complemented only some of the Nrf− mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c 552 synthesis in this region. Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c 552′ for the attachment of the haem group to the apocytochrome and for cytochrome c 552 export into the periplasm.
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Two-membered mixed cultures of methanogenic and aerobic bacteria in O2-limited chemostats
More LessSUMMARY: Three different co-cultures composed of a methanogenic and a strictly aerobic bacterium were grown under O2-limitation in continuous cultures. The combinations used were (1) Methanobacterium formicicum with the aerobic heterotroph Comamonas testosteroni; (2) M. formicicum with a methanotrophic Methylocystis species; and (3) Methanosavcina barkeri with C. testosteroni. Although true steady-states were not obtained, growth and metabolic activity of the methanogenic and aerobic organisms occurred during O2-limited growth of these mixed cultures over extended periods of time. Co-cultures with C. testosteroni were considerably more stable than those with Methylocystis. Co-cultures with M. barkeri were less O2-sensitive than those with M. formicicum. C. testosteroni exhibited a higher O2-affinity than Methylocystis, resulting in a lower dissolved oxygen tension and a superior protection of the methanogenic bacteria against O2-poisoning than in mixed cultures with Methylocystis. The dissolved O2-concentrations in the mixed cultures were below the detection limit of the O2-probes used (0.2 μM). Calculations based on growth properties of pure cultures of C. testosteroni, M. barkeri and M. formicicum suggested that the dissolved O2-concentrations in the mixed cultures, as well as the O2-inhibition constants (apparent K 1 o2) of the methanogens were in the nanomolar range.
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Growth inhibition and pyruvate overflow during glucose metabolism of Eubactevium limosum are related to a limited capacity to reassimilate CO2 by the acetyl-CoA pathway
More LessSUMMARY: The growth characteristics of Eubactevium limosum, an anaerobic acetogen, have been described kinetically for fermentation of glucose. A short-lived exponential growth phase was associated with a homoacetogenic fermentation in which all catabolic CO2 liberated was reassimilated via the acetyl-CoA pathway. During this phase no additional products were observed. A major shift in the metabolism leading to a mixed-acid fermentation pattern (acetate and butyrate) occurred coincident with the onset of the linear growth phase. Significantly diminished yields of acetate in this decelerating growth period were attributed to the limited capacity of the enzymes responsible for reductive generation of methyl donor within the acetyl-CoA pathway: CO2 and H2 accumulated in the gas phase. Pyruvate overflow (and to a lesser extent lactate, glycerate and carbon monoxide accumulation) were also observed, though pyruvate-ferredoxin oxidoreductase specific activity remained constant. In all experiments specific biomass formation rates were directly related to the rate of acetyl-CoA formation. The exponential growth phase was prolonged and the production of all compounds other than acetate was diminished when the medium was supplemented with tungsten, suggesting that formate dehydrogenase may be the enzyme limiting the correct functioning of the acetyl-CoA pathway.
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The effect of salinity and compatible solutes on the biosynthesis of cyclopropane fatty acids in Pseudomonas halosaccharolytica
More LessSUMMARY: The moderately halophilic eubacterium Pseudomonas halosacchavolytica has been grown at salinities over the range 5-25 % (w/v), equivalent to 0.7-3.5 M-NaCl, and the fatty acid composition determined in the late-exponential and stationary phases of batch culture. There was an increase in the proportion of cyclopropane fatty acids (CFA) as the cultures went into stationary phase at all salinities; the overall proportion of CFA was higher in the media containing more salt. The biosynthesis of CFA in P. halosacchavolytica was determined using radiolabelled S-adenosylmethionine as the precursor incubated in cell-free extracts prepared by breaking bacteria with a French press. Compared with the activity obtained in 100 mM-phosphate buffer, the activity of CFA synthetase was inhibited by the addition of NaC1 or KC1, but stimulated up to 12-fold by added glycinebetaine, with maximum activity at 3 M. Although the specific activity of CFA synthetase in lysates from cultures grown in 0.7 or 2.1 M-NaC1 were similar in the presence of 3 M-glycinebetaine, the enzyme activity in low-salinity cultures was better adapted to function in 1 M-glycinebetaine. Shift-up experiments, in which CFA synthetase activity was assayed in cell-free extracts prepared at different times after increasing culture salinity from 0.7 to 2.1 M-NaC1, showed that the activity of the enzyme was immediately responsive to compatible solute concentration changes and indicated that enzyme induction would not be required to achieve the salt-dependent alterations in membrane lipid CFA composition in vivo. A range of other compatible organic solutes stimulated CFA synthetase activity to a much lesser extent (1.8-fold) compared with glycinebetaine. It is suggested that a compatible solute, which is normally accumulated during osmo(halo)adaptation by an organism in order to contribute towards osmotic balance, does not behave passively towards intracellular proteins but can also stimulate enzyme activity.
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Cell wall assembly in Staphylococcus aureus: proposed absence of secondary crosslinking reactions
More LessSUMMARY: The distribution of muropeptides formed by muramidase digestion of peptidoglycan from Staphylococcus aureus H was determined by gel-filtration HPLC. The observed crosslinking pattern supports the conclusion that incorporation of peptidoglycan in S. aureus proceeds by a similar mechanism to that proposed earlier for Bacillus megaterium. In this mechanism single glycan-peptide strands are incorporated into the sacculus by crosslinking reactions that take place only between the monomer muropeptide units of the incoming glycopeptide and muropeptides present in the innermost region of wall at the wall-membrane interface: such crosslinking reactions take place only during incorporation and no other crosslinking reactions occur. This assembly process has now been termed restricted monomer addition. The present analysis shows that the distribution of muropeptides in S. aureus peptidoglycan is in excellent agreement with that predicted by this mechanism. We propose that cell wall assembly in S. aureus proceeds via restricted monomer addition without any requirement for the secondary crosslinking reactions that have been suggested to occur in this organism. The high degree of crosslinking in S. aureus, 80% in this study, may result mainly from the freedom for crosslinking provided by the pentaglycine bridge peptide.
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Incorporation and fate of N-acetyl-D-glucosamine during hyphal growth in Streptomyces
More LessSUMMARY: N-AcetyI-D-[1-14C]glucosamine ([14C]GlcNAc) was used to label specifically the cell wall in Streptomyces antibioticus. Lysozyme solubilized 86.5% of the radioactivity present in the trichloroacetic acid-precipitated and RNAase plus trypsin-digested fraction of hyphae pulse-labelled with [14C]GlcNAc for 3 min in a medium containing glucose, yeast extract and a mixture of nine amino acids. Experiments with [14C]GlcNAc-labelled hyphae revealed that growth of Streptomyces occurs without turnover of peptidoglycan. Autoradiographic analysis of S. antibioticus hyphae indicated that cell wall elongation occurs by intercalation of newly synthesized wall polymers at the tip, but also over a relatively broad zone that, depending on the hyphal length, extends up to 6-10 μm from the tip.
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Biochemical analysis of germination mutants to characterize germinant receptors of Bacillus subtilis 1604 spores
More LessSUMMARY: Spores of Bacillus subtilis 1604 can be induced to germinate by incubation in L-Ala (the ALA pathway) or in a combination of β-D-gIucose (Glc), β-fructose (Fru), L-Asn and K+ (the GFAK pathway). Biochemical analysis of the germination response of a gerA mutant deficient in the ALA pathway revealed that L-Ala can replace L-Asn in the GFAK pathway (the GFAlaK pathway). In contrast to the ALA pathway of both the wild-type and of a gerB mutant, the GFAlaK pathway w as insensitive to D-Ala and showed the same overall inhibitor profile as the GFAK pathway of wild-type and gerA spores. It is deduced that a second L-Ala receptor with different characteristics to that functioning in the ALA pathway is present in wild-type spores. Analysis of the germination response of a gerB mutant showed that whilst the rate of ALA germination could be stimulated by Glc as well as by Fru in the presence of Glc, the spores could not germinate in GFAK. In addition, Glc and Fru were unable to reverse D-Ala inhibition of L-Ala germination which they do in the wild-type. Thus, in the gerB mutant, the L-Ala/L-Asn receptor in the GFAK pathway is defective. It is concluded that the germination receptors in the ALA and GFAK pathways can functionally interact with each other to initiate B. subtilis spore germination. This conclusion is discussed in relation to proposed models of triggering of spore germination.
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- Systematics
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Gellan-related polysaccharides and the genus Sphingomonas
More LessSUMMARY: The biochemical and physiological characteristics of several different existing bacterial isolates which secrete gellan-related polysaccharides were compared. Although they were originally classified into diverse genera, these bacteria are shown here to be closely related to each other and to members of the genus Sphingomonas.
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