- Volume 142, Issue 5, 1996
Volume 142, Issue 5, 1996
- Pathogenicity And Medical Microbiology
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In vitro phagocytosis and survival of Streptococcus suis capsular type 2 inside murine macrophages
More LessIn this study, data on phagocytosis of Streptococcus suis and its survival inside macrophages are presented. Mouse peritoneal macrophages were incubated in the presence of one of five different strains of S. suis capsular type 2: a virulent wild-type strain (1591), a non-capsulated non-virulent mutant strain (M2), a poorly capsulated non-virulent mutant strain (M42), a non-virulent capsulated strain (1330), and the wild-type reference (virulent) strain 5735. Opsonized or non-opsonized bacteria were incubated with macrophages in vitro and samples were obtained after 1 and 3 h incubation. Phagocytosis as well as live and dead intracellular organisms were determined by acridine orange and crystal violet staining. After 1 h incubation, non-opsonized virulent and non-virulent capsulated bacteria were poorly phagocytosed (by less than 7% of the macrophages), whereas the non-capsulated non-virulent mutant strain was highly phagocytosed (by more than 68% of the macrophages). The M42 mutant strain was more phagocytosed than the capsulated strains but less than the non-capsulated M2 mutant strain (35%). In contrast, a higher percentage of live bacteria was observed inside macrophages for the capsulated strains (1591 and S735) than for the non- or poorly capsulated mutant strains (M2 and M42). Opsonization of bacteria with rabbit serum or heat-inactivated rabbit serum significantly increased phagocytosis. For every opsonized strain, after 3 h incubation, the percentage of live bacteria within macrophages was considerably lower than the corresponding non-opsonized strains. In conclusion, the capsule of S. suis type 2 appears to act as an important anti-phagocytic factor. However, virulent capsulated non-opsonized strains can be phagocytosed by mouse peritoneal macrophages within which they appear to survive for at least 3 h. Serum factors other than complement increase not only phagocytosis but also intracellular killing of S. suis of both capsulated and non-capsulated strains.
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- Physiology And Growth
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Asparagine degradation in Rhizobium etli
More LessThe degradation of asparagine by Rhizobium etli involves asparaginase and aspartate ammonia-lyase (L-aspartase). The two enzymes were shown to be positively regulated by asparagine and negatively regulated by the carbon source. Asparaginase activity was not regulated by oxygen concentration or by nitrogen catabolite repression. Induction of both enzymes by asparagine enables R. etli to utilize asparagine as carbon source. Asparaginase may also be involved in maintaining the optimal balance between asparagine and aspartate. Aspartase was not involved in the utilization of aspartate or glutamate as carbon source. The presence of high levels of the two enzymes in R. etli bacteroids suggests that they may have a role in symbiosis between R. etli and Phaseolus vulgaris.
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Acquisition of iron by the non-siderophore-producing Pseudomonas fragi
More LessThe iron requirement, siderophore production and iron uptake mechanisms of the type strain Pseudomonas fragi ATCC 4973 and five P. fragi isolates from meat were analysed. The strains exhibited a high sensitivity to iron starvation: their growth was strongly inhibited in medium supplemented with the iron chelator ethylenediamine di(hydroxyphenylacetic acid) or in medium treated with 8-hydroxyquinoline to remove contaminating iron. No siderophores were detectable in the growth supernatants of iron-starved cells. Cross-feeding experiments in iron-depleted medium showed, however, that the bacterial growth could be strongly stimulated by siderophores of foreign origin including desferriferrioxamine B, enterobactin and some pyoverdines. Moreover, all the strains were capable of efficiently using the iron sources present in their natural environment, i.e. transferrin, lactoferrin and haemoglobin. Iron starvation led to the specific production of supplementary outer-membrane proteins of apparent molecular mass ranging from 80 to 88 kDa. Furthermore, growth in the presence of exogenous siderophores resulted, in some strains, in the induction of siderophore-mediated iron uptake systems. For one strain the concomitant synthesis of an iron-regulated, siderophore-inducible outer-membrane protein was observed.
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Cell density influences antibiotic biosynthesis in Streptomyces clavuligerus
More LessProduction of cephamycin C and clavulanic acid by Streptomyces clavuligerus took place during the exponential phase of growth in a defined medium. Both antibiotic biosynthetic pathways were activated shortly after spore germination, but the timing and kinetics of activation were affected by inoculum density. Rapid activation was favoured by high inoculum density or by growth in medium conditioned by previous incubation of S. clavuligerus spores or mycelium. A heat-resistant conditioning factor able to accelerate the acquisition of antibiotic-biosynthetic capacity when added to low-density cultures was released in suspensions of spores in water. Conditioning factor was also obtained in suspensions of spores from different Streptomyces species or of Bacillus cells, indicating that the signal was not produced specifically by S. clavuligerus. Fractionation of conditioning factor showed that its effect was not due to a single molecule. The fractions contained amino acids (as free amino acids and oligopeptides) in amounts that roughly correlated with their respective conditioning power. Furthermore, the conditioning effect was reproduced by supplementing defined medium with amino acids and peptides in concentrations that mimicked those found in conditioning factor. When individually tested at concentrations in the micromolar range, only some amino acids were able to stimulate antibiotic biosynthetic capacity. This stimulation was also promoted by low concentrations (less than 1 μg ml-1) of peptide mixtures obtained with different proteolytic enzymes. The results suggest that both amino acids and peptides are responsible for the effects of conditioning factor released by spores. Possible implications of intercellular signalling on activation of secondary metabolism are discussed.
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Metabolism of glycoprotein-derived sialic acid and N-acetylglucosamine by Streptococcus oralis
More LessNine strains of Streptococcus oralis, isolated from blood cultures of patients with infective endocarditis or from the oral cavity as part of the normal flora, were examined for their ability to elaborate sialidase (neuraminidase) and N-acetylglucosaminidase, enzymes which are involved in the degradation of glycoproteins. Both glycosidases were induced when bacteria were grown in a minimal medium supplemented with porcine gastric mucin, a model glycoprotein, and repressed when growth occurred in the presence of glucose. Cell-free extracts of mucin-grown cultures expressed elevated levels of N-acetylneuraminate pyruvate-lyase (the first intracellular enzyme in the pathway of N-acetylneuraminate catabolism), N-acetylglucosamine (GlcNAc)-6-phosphate deacetylase and glucosamine-6-phosphate deaminase (enzymes involved in the intracellular catabolism of GlcNAc 6-phosphate); activity of each of these intracellular enzymes was markedly repressed when bacteria were grown in the presence of glucose. Three strains of S. oralis were also grown in media supplemented with α1-acid glycoprotein, a major component of human plasma. Cells from these cultures expressed high levels of sialidase, N-acetylglucosaminidase, and the intracellular enzymes involved in the catabolism of N-acetyl-sugars released by the action of these glycosidases. High-resolution 1H-NMR spectroscopy of spent culture supernatants revealed that sialic acid and GlcNAc residues of the molecularly mobile oligosaccharide side-chains of α1-acid glycoprotein had been hydrolysed and the released sugars internalized by the bacteria. These data indicate that S. oralis has the ability to hydrolyse constituents of oligosaccharide side-chains of host-derived glycoproteins and to utilize simultaneously these released carbohydrates. The biochemical characteristics induced by the growth of S. oralis on glycoproteins may play a role in the survival and persistence of these bacteria at the infection site in vivo.
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The role of the outer membrane in formaldehyde tolerance in Escherichia coli VU3695 and Halomonas sp. MAC
More LessTo investigate the mechanism of formaldehyde tolerance in Gram-negative bacteria, two formaldehyde-tolerant strains, Escherichia coli VU3695 and Halomonas sp. MAC (DSM 7328), and formaldehyde-sensitive revertants obtained by ethidium bromide or novobiocin treatment were studied. The presence of high levels of formaldehyde dehydrogenase activity alone proved insufficient to confer tolerance to high formaldehyde concentrations, as shown by the high activity displayed by formaldehyde-sensitive revertants of Halomonas MAC. Moreover, formaldehyde-tolerant strains also proved to be tolerant to high concentrations of acetaldehyde and glutaraldehyde, which are not oxidized by formaldehyde dehydrogenase. Treatment with sublethal concentrations of EDTA rendered the resistant strains highly sensitive to formaldehyde without affecting the activity of formaldehyde dehydrogenase. Comparison of the outer membrane proteins of formaldehyde-resistant strains with those of their sensitive revertants showed the presence of at least one additional high molecular mass protein in the tolerant strains. It is concluded that formaldehyde tolerance in the bacteria studied depends on the composition and structure of the outer membrane.
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Complementary chromatic adaptation alters photosynthetic strategies in the cyanobacterium Calothrix
More LessThe cyanobacterium Calothrix sp. strain PCC 7601 drastically changes phycobiliprotein composition and colour in response to light quality, through complementary chromatic adaptation (CCA). Red light promotes phycocyanin-II and inhibits phycoerythrin synthesis, while green light has the opposite effect, through changes in transcription regulated by a putative green/red photoreceptor(s). The effects of CCA on photosynthesis were characterized by measuring oxygen evolution and chlorophyll fluorescence parameters. Cells fully acclimated to either red or green light achieve a similar photosynthetic quantum yield of oxygen evolution (light-use efficiency). Shifting acclimated cells from green to red or from red to green light caused similar 40% drops in photosynthetic quantum yield. Therefore, full CCA significantly increases light use efficiency, which is of great importance under light-limited growth. Cells growing under red light are in state I, with very low PS II to PS I energy transfer, since red light is absorbed both by phycocyanin in the phycobilisome/PS II supracomplex and by PS I chlorophyll. Cells growing under green light are in state II, with high transfer of excitation energy from the phycobilisome/PS II supracomplex to PS I. This transfer allows green light captured by phycoerythrin to ultimately drive both PS I and PS II photochemistry.
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- Plant-Microbe Interactions
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Genetic relationships among rhizopine-producing Rhizobium strains
More LessChromosomal and symbiosis-related genotypes of rhizopine-producing and non-producing isolates of Rhizobium meliloti and Rhizobium leguminosarum were examined by multilocus enzyme electrophoresis and RFLP. The distribution of rhizopine production in both species was found to be independent of host genotype. Conversely, rhizopine production was associated with particular symbiotic plasmid types. This association may explain the observed distribution of rhizopine production in R. leguminosarum and R. meliloti. Rhizopine synthesis (mos) genes showed greater sequence divergence than rhizopine catabolism (moc) genes in both R. meliloti and R. leguminosarum. Furthermore, mos and moc genes were less divergent in R. leguminosarum than R. meliloti, suggesting a more recent evolution in the former species.
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Nitrogen limitation of chemostat-grown Rhizobium etli elicits higher infection-thread formation in Phaseolus vulgaris
More LessThe symbiotic association between rhizobia and legume roots is a complex process involving many steps. An infection thread is a tubular structure of host origin formed during the infection of legume roots by rhizobia. Previous studies with batch cultures have reported that optimal attachment of rhizobia to root hairs coincides with nutrient limitation. In this study, the ability of chemostat-grown, nutrient-limited Rhizobium etli cells to form infection threads with its symbiotic partner Phaseolus vulgaris was investigated. Rhizobia were grown in a chemostat in synthetic media under C- or N-limiting conditions. Infection-thread formation was examined after inoculation of seedlings with a rhizobial cell suspension from each treatment. The number of infection threads was estimated by light microscopy after staining root sections with o-toluidine. Exopolysaccharide (EPS) production was also measured, and the cellular content and electrophoretic pattern of lipopolysaccharide (LPS) determined semiquantitatively. N-limited cells showed a markedly higher infectivity (measured as infection-thread formation) than C-limited cells. With one of the two bean cultivars used, the number of infection threads produced by N-limited cells was higher than that produced by exponentially growing cells in batch cultures. The higher infectivity of N-limited cells was correlated with higher EPS production. Electrophoretic analysis of LPS showed that C- and N-limited cells shared a common profile but the relative concentration of short LPS forms differed.
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Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4
The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthesized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.
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Genetic transfer of amylovoran and stewartan synthesis between Erwinia amylovora and Erwinia stewartii
DNA fragments with ams genes of Erwinia amylovora and cps genes of Erwinia stewartii were transferred to exopolysaccharide (EPS)-deficient mutants of the other species. The resulting EPSs were characterized by sensitivity to EPS-dependent bacteriophages, staining with amylovoran-specific fluorescein-isothiocyanate-labelled lectin and chemical techniques, such as determination of the sugar composition and methylation analysis in order to distinguish between amylovoran and stewartan. Degradation by the stewartan-dependent phage ø-K9 was used to detect stewartan production, and staining with a lectin from Abrus precatorius detected amylovoran capsules. The patterns of sugar linkages were determined by methylation analysis. Stewartan contained a significantly higher glucose to galactose ratio than amylovoran and produced a characteristic signal from 6-linked glucose residues. By these criteria, most E. stewartii cps mutants displayed exclusively amylovoran synthesis when complemented with the ams cluster, and E. amylovora ams mutants complemented with E. stewartii cps genes produced stewartan. The complementation to an EPS-positive phenotype may require most genes of the ams or the cps operon. An exception was an E. stewartii cpsK mutant that made predominantly stewartan when complemented with the ams cosmid. IR spectra showed that both amylovoran and stewartan were acylated when synthesized in E. amylovora, but not in E. stewartii. The amylovoran-producing E. stewartii merodiploids regained virulence to corn seedlings when mucoidy was restored by the ams cluster, but the stewartan-producing E. amylovora ams-lcps + strains were weakly virulent on pear slices and avirulent on apple seedlings.
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- Corrigendum
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Volume 170 (2024)
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