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Microbiology Society logo - Volume 146, Issue 11, 2000

Volume 146, Issue 11

Volume 146, Issue 11, 2000

  • Pathogenicity And Medical Microbiology

      • The regulon is involved in the opportunistic properties of and in mice and insects

        https://doi.org/10.1099/00221287-146-11-2825

        has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous Cry and strains were investigated in an insect species, , and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, co-ingestion of 10 spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if spores of the Δ mutant strain were used. In mice, nasal instillation of 10 spores of the parental strain caused 100% mortality whereas instillation with the same number of Δ strain spores caused much lower or no mortality Similar effects were obtained if vegetative cells were used instead of spores. The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of and , using sheep, horse and human erythrocytes and haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in Δ strains. Together, the results indicate that the pathogenicity of strain 407 and strain ATCC 14579 is controlled by PlcR.

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  • Physiology And Growth

      • Involvement of the locus in core oligosaccharide and O polysaccharide assembly in

        https://doi.org/10.1099/00221287-146-11-2803

        -Rhamnose (-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of . In this study, four contiguous genes () responsible for the synthesis of dTDP--Rha in have been cloned and characterized. Non-polar chromosomal mutants were generated in strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting. mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen. A PAO1 double mutant (deficient in biosynthesis of both -Rha and -Rha) was constructed to facilitate structural analysis of the mutant core region. This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen. These results provide the genetic and structural evidence that -Rha is the receptor on the LPS core for the attachment of O polysaccharides. This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.

    • A novel thermostable multidomain 1,4-β-xylanase from ‘’ and effect of its xylan-binding domain on enzyme activity

      https://doi.org/10.1099/00221287-146-11-2947

      The GenBank accession number for the sequence reported in this paper is AF200304.

      The nucleotide sequence of the complete gene, encoding a novel multidomain xylanase XynA of ‘’, was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 °C (XynAd1/2) to 60 °C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1,4-β-xylanases, but differ in their ability to cleave small xylooligosaccharides.

    • A periplasmic, α-type carbonic anhydrase from is essential for bicarbonate uptake

      https://doi.org/10.1099/00221287-146-11-2957

      Intact cells of the purple non-sulfur bacterium growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified >2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene () was cloned and its sequence revealed that it was homologous to α-type CAs. The upstream region of was fused to the gene and β-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC in the range of 10 M, and in the culture media concentrations as low as 30 μM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An :: mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO and that there is no expression under aerobic conditions.

    • High hopanoid/total lipids ratio in mycelia is not related to the nitrogen status

      https://doi.org/10.1099/00221287-146-11-3013

      The GenBank/EMBL/DDBJ accession numbers for the sequences in this paper are AJ251388–91 and AJ251393–4.

      Vesicles are specific structures which are produced under nitrogen-limiting culture conditions. Hopanoids are the most abundant lipids in these vesicles and are believed to protect the nitrogenase against oxygen. The amounts and quality of each hopanoid were estimated in different strains cultivated under nitrogen-depleted and nitrogen-replete conditions in order to detect a possible variation. Studied strains nodulating were phylogenetically characterized by analysis of the intergenic region as closely related to genomic species 4 and 5. Phylogenetically different strains belonging to three infectivity groups were cultivated in the same medium with and without nitrogen source for 10 d before hopanoid content analysis by HPLC. Four hopanoids together accounted for 23–87% and 15–87% of the total lipids under nitrogen-replete and nitrogen-depleted culture conditions, respectively. Two of the hopanoids found, bacteriohopanetetrols and their phenylacetic acid esters, have previously been described in . Two new hopanoids, moretan-29-ol and a bacteriohopanetetrol propionate, have also been identified. The moretan-29-ol and bacteriohopanetetrols were found to be the most abundant hopanoids whereas the bacteriohopanetetrol propionate and phenylacetates were present at a concentration close to the limit of detection. The ratio of (bacteriohopanetetrols + moretan-29-ol)/(total lipids) varied in most of the strains between nitrogen-depleted and nitrogen-replete culture conditions. In most of the strains, the hopanoid content was found to be slightly higher under nitrogen-replete conditions than under nitrogen-depleted conditions. These results suggest that remobilization, rather than neosynthesis of hopanoids, is implicated in vesicle formation in under nitrogen-depleted conditions.

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  • Plant-Microbe Interactions

      • The bv. gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation

        https://doi.org/10.1099/00221287-146-11-2987

        The GenBank accession number for the sequence reported in this paper is AF155830.

        A bv. VF39 gene () encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation () system, was identified, cloned and characterized. The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the gene products of other bacterial species. Downstream of the VF39 resides an open reading frame with similarity to the virulence factor gene . Mutation of the gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine. In addition, neither uridylylation of P nor induction of the -regulated gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains. This strongly suggests that mutants harbour a permanently deuridylylated P protein and as a consequence are unable to activate transcription from NtrC-dependent promoters. The gene itself is expressed constitutively, irrespective of the nitrogen content of the medium. A functional GlnD protein is not essential for nitrogen fixation in bv. , but detection of expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the system functions in symbiosis. The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5′-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain.

    • Phylogenetic diversity of rhizobial strains nodulating L.

      https://doi.org/10.1099/00221287-146-11-2997

      The EMBL accession numbers for the sequences reported in this paper are AJ271898–AJ271902 for the strains Rob6, Rob8 and Rob23 and the strains Rob18 and Rob20, respectively.

      Lack of knowledge exists regarding the diversity of rhizobial strains nodulating black locust ( L.), which is a neophytic tree species widely distributed in Europe. Seventeen rhizobial strains isolated from nodules of black locust at a German location were examined by phenotypic characterization and 16S rDNA analysis. The isolates were classified in nine 16S rDNA genotypes using a set of seven endonucleases. Based on RFLP analysis and sequencing, the strains were shown to belong to the genera (76%) and (24%). Five genotypes were identical to the species , , , and . A strong similarity between the 16S rDNA sequence of another two genotypes and (999%) as well as the strain R88b (998%) was found. The two remaining genotypes were classified in the genus , without a significant relationship at the species level. Comparing isolates nodulating and , a parallel picture of phylogenetic diversity was detected with a range of phylogenetically different rhizobia and dominating. For this study, 18 rhizobial strains which had originally been isolated from a forest in Maryland where black locust is native were additionally analysed. Results revealed seven genotypes all belonging to the genus , with four genotypes identical to the isolates from the German sampling location. Whereas the genotype identical to dominated within the strains obtained from the German location, the dominance of a genotype identical to was found among the strains from the native location. Summarizing data from both locations, was nodulated with various genomic species, most of which belonged to the genus . Concerning phenotypic features such as growth rate, pH tolerance or use of certain carbohydrates, most isolates corresponded to described species and genera. However, there were differences in salt tolerance between these isolates and the corresponding reference strains. Overall, the results demonstrated a high phenotypic and phylogenetic diversity of rhizobial strains nodulating . This may be a characteristic of neophytic and other widely spread legumes and may contribute to the success of black locust as a pioneer tree species for the temperate zone.

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  • Systematics And Evolution

      • Comparative sequence analyses reveal frequent occurrence of short segments containing an abnormally high number of non-random base variations in bacterial rRNA genes

        https://doi.org/10.1099/00221287-146-11-2845

        The GenBank accession numbers for the 23S rRNA sequences determined in this study are AF192136–AF192150.

        rRNA genes are thought unlikely to be laterally transferred, because rRNA must coevolve with a large number of cellular components to form the highly sophisticated translation apparatus and perform protein synthesis. In this paper, the authors first hypothesized that lateral gene transfer (LGT) might occur to rRNA genes via replacement of gene segments encoding individual domains of rRNA: the ‘simplified complexity hypothesis’. Comparative sequence analyses of the 16S and 23S rRNA genes from a large number of actinomycete species frequently identified rRNA genes containing short segments with an abnormally high number of non-random base variations. These variations were nearly always characterized by complementing covariations of several paired bases within the stem of a hairpin. The nature of these base variations is not consistent with random mutations but satisfies well the predictions of the ‘simplified complexity hypothesis’. The most parsimonious explanation for this phenomenon is the lateral transfer of rRNA gene segments between different bacterial species. This mode of LGT may create mosaic rRNA genes and occur repeatedly in different regions of a gene, gradually destroying the evolutionary history recorded in the nucleotide sequence.

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