- Volume 146, Issue 5, 2000
Volume 146, Issue 5, 2000
- Physiology And Growth
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Metabolism of carveol and dihydrocarveol in Rhodococcus erythropolis DCL14
More LessRhodococcus erythropolis DCL14 assimilates all stereoisomers of carveol and dihydrocarveol as sole source of carbon and energy. Induction experiments with carveol- or dihydrocarveol-grown cells showed high oxygen consumption rates with these two compounds and with carvone and dihydrocarvone. (Dihydro)carveol-grown cells of R. erythropolis DCL14 contained the following enzymic activities involved in the carveol and dihydrocarveol degradation pathways of this micro-organism: (dihydro)carveol dehydrogenase (both NAD+- and dichlorophenolindophenol-dependent activities), an unknown cofactor-dependent carvone reductase, (iso-)dihydrocarvone isomerase activity, NADPH-dependent dihydrocarvone monooxygenase (Baeyer–Villiger monooxygenase), ϵ-lactone hydrolase and an NAD+-dependent 6-hydroxy-3-isopropenylheptanoate dehydrogenase. Product accumulation studies identified (4R)-carvone, (1R,4R)-dihydrocarvone, (4R,7R)-4-isopropenyl-7-methyl-2-oxo-oxepanone, (3R)-6-hydroxy-3-isopropenylheptanoate, (3R)-3-isopropenyl-6-oxoheptanoate, (3S,6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone and (5R)-6-hydroxy-5-isopropenyl-2-methylhexanoate as intermediates in the (4R)-carveol degradation pathway. The opposite stereoisomers of these compounds were identified in the (4S)-carveol degradation pathway. With dihydrocarveol, the same intermediates are involved except that carvone was absent. These results show that R. erythropolis DCL14 metabolizes all four diastereomers of carveol via oxidation to carvone, which is subsequently stereospecifically reduced to (1R)-(iso-)dihydrocarvone. At this point also dihydrocarveol enters the pathway, and this compound is directly oxidized to (iso-)dihydrocarvone. Cell extracts contained both (1R)-(iso-)dihydrocarvone 1,2-monooxygenase and (1S)-(iso)-dihydrocarvone 2,3-monooxygenase activity, resulting in a branch point of the degradation pathway; (1R)-(iso-)dihydrocarvone was converted to 4-isopropenyl-7-methyl-2-oxo-oxepanone, while (1S)-(iso)-dihydrocarvone, which in vivo is isomerized to (1R)-(iso-)dihydrocarvone, was converted to 6-isopropenyl-3-methyl-2-oxo-oxepanone. 4-Isopropenyl-7-methyl-2-oxo-oxepanone is hydrolysed to 6-hydroxy-3-isopropenylheptanoate, which is subsequently oxidized to 3-isopropenyl-6-oxoheptanoate, thereby linking the (dihydro)carveol degradation pathways to the limonene degradation pathway of this micro-organism. 6-Isopropenyl-3-methyl-2-oxo-oxepanone is, in vitro, hydrolysed to 6-hydroxy-5-isopropenyl-2-methylhexanoate, which is thought to be a dead-end metabolite.
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Rhodococcus opacus strain PD630 as a new source of high-value single-cell oil? Isolation and characterization of triacylglycerols and other storage lipids
More LessThe triacylglycerol (TAG)-accumulating, hydrocarbon-degrading bacterium Rhodococcus opacus strain PD630 and chemically induced storage-deficient mutants derived from this strain were investigated for their capability to accumulate storage lipids in the cytoplasm during cultivation under nitrogen-limiting conditions. Acylglycerols were analysed by matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI–TOF) and by reversed-phase HPLC. Fatty acids comprising 13–19 carbon atoms in various acylglycerols constituted up to 76% of the cellular dry weight in gluconate-grown cells, with a significant proportion of odd-numbered fatty acids. Hydrolysis using pancreatic lipase and deacylation with ethyl magnesium bromide were employed to identify the stereospecific distribution of fatty acids at the glycerol. This analysis showed that the fatty acids were not randomly distributed between the three positions of the glycerol backbone. In comparison with common plant fats, where the longer and higher unsaturated fatty acids are predominantly found at position 2, R. opacus PD630 accumulated only the shorter and saturated fatty acids in this position. More than 100 mutants accumulating TAG at a significantly lower rate were obtained by chemical mutagenesis and identified by staining with Sudan Black B. All the mutants showed similar neutral lipid patterns by TLC analysis, with a small distinct spot exhibiting the same R F value as TAG; this was identified as a residual amount of TAG by preparative TLC and MALDI-TOF, indicating that this bacterium is possibly capable of synthesizing TAGs by at least two different pathways.
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- Plant-Microbe Interactions
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The exbD2 gene as well as the iron-uptake genes tonB, exbB and exbD1 of Xanthomonas campestris pv. campestris are essential for the induction of a hypersensitive response on pepper (Capsicum annuum)
More LessThe tonB, exbB and exbD1 genes of Xanthomonas campestris pv. campestris are essential for ferric iron uptake. In contrast, the exbD2 gene located in the same gene cluster is not essential. Mutational analysis revealed that the ferric-iron-uptake genes tonB, exbB and exbD1 are necessary for the induction of a hypersensitive response (HR) on the nonhost plant pepper (Capsicum annuum) and the induction of typical black rot symptoms on the host plant cauliflower (Brassica oleracea). Again, the exbD2 gene behaved differently. It was found to play a role only in the induction of the HR in pepper but not in the induction of black rot symptoms in cauliflower. Due to the low iron concentration in the plant tissue, the titre of viable bacteria of the ferric-iron-uptake mutants tonB, exbB and exbD1 decreased after leaf infiltration of pepper. The exbD2 mutant, however, which is not impaired in ferric iron uptake, multiplied in the pepper leaf tissue and grew even better than the wild-type strain, probably due to its failure to induce the HR. Nevertheless, the tonB, exbB and exbD1 mutant strains were able to spread systemically in cauliflower.
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- Systematics And Evolution
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Two distinct 18S rRNA secondary structures in Dipodascus (Hemiascomycetes)
More LessThe DDBJ accession numbers for the sequences reported in this paper are shown in Table 1 T1 .
The nucleotide sequences of the 18S rRNA gene from ascomycetous yeast-like fungi in the genera Dipodascus, Galactomyces and Geotrichum were determined and the tested strains were separated into two groups by sequence length. In group 1, the length and secondary structure of 18S rRNA corresponded to those of typical eukaryotes. In group 2, the 18S rRNA gene sequences were about 150 nt shorter than those of most other eukaryotes and the predicted secondary structure lacked helices 10 and E21-5. Many substitutions and some deletions in group 2 18S rRNA gene were not only found in variable regions, but also in regions that are highly conserved among ascomycetes. Despite the considerable differences in 18S rRNA gene sequence and secondary structure between group 2 and other fungi, including group 1, phylogenetic analysis revealed that groups 1 and 2 are closely related. These findings suggest that a number of deletions occurred in the 18S rRNA of the common ancestor of group 2 strains.
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Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis
The GenBank accession number for the sequence reported in this paper is AF177275.
Free-living amoebae are increasingly being recognized to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a variety of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeba endocytobionts constituting the recently proposed family Parachlamydiaceae are of special interest as potential human pathogens. In this study coccoid bacterial endocytobionts of a Hartmannella vermiformis isolate were analysed. Infection of H. vermiformis with these bacteria resulted in prevention of cyst formation and subsequent host-cell lysis. Transfection experiments demonstrated that the parasites were not capable of propagating within other closely related free-living amoebae but were able to infect the distantly related species Dictyostelium discoideum. Electron microscopy of the parasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts, in contrast to Chlamydia species, do not reside within a vacuole. Comparative 16S rRNA sequence analysis showed that the endocytobiont of H. vermiformis, classified as Neochlamydia hartmannellae gen. nov., sp. nov., is affiliated to the family Parachlamydiaceae. Confocal laser scanning microscopy in combination with fluorescence in situ hybridization using rRNA-targeted oligonucleotide probes confirmed the intracellular localization of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge of the phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoebae of the genus Hartmannella.
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Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes
More LessThe GenBank accession numbers for the sequences determined in this work are AF197508–AF197534.
Ninety-three Bacteroides fragilis strains of different origin were analysed by multilocus enzyme electrophoresis (MLEE). Fourteen of the 15 genetic loci analysed were polymorphic, whilst nucleoside phosphorylase was monomorphic. There was a mean of six alleles per locus and a mean genetic diversity of 0·393. Cluster analysis identified 90 electrophoretic types (ETs) separated into two major phylogenetic divisions at a genetic distance of 0·70. Division I consisted of 81 ETs carrying the endogenous class A β-lactamase gene cepA, whereas division II comprised 9 ETs carrying the class B β-lactamase gene cfiA, but not cepA. The presence of these two genes was assessed by PCR and the expression of the cfiA gene was investigated by determining the level of resistance to the antibiotic imipenem. MLEE showed a smaller genetic distance among the genotypes of the imipenem-resistant than among the imipenem-susceptible strains. No other particular cluster was observed. The enterotoxin gene (bft) was detected by PCR: DNA sequencing of the products obtained showed that the different bft alleles (bft-1, bft-2 and bft-3) were scattered randomly troughout the phylogenetic tree. No association between distinct clones and clinical manifestations (sepsis, abscesses, diarrhoea), geographical origin or host origin (human or animal) could be found.
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