Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I. Ultrastructure and preliminary chemical analysis.
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The distribution of polysaccharides from the seed cell walls of 57 samples of Vochysiaceae native to Brazil were studied, comprising 16 species distributed among the genera Callisthene, Qualea, Salvertia and Vochysia. The polysaccharides were extracted with hot water, then hydrolyzed with the resulting monomers analyzed by HPLC. All samples yielded arabinose, galactose, glucose. mannose and rhamnose, the relative amounts of each monomer, however, varying from one sample to another. Arabinose was always the predominant component, which implies that it might possibly be used as a marker of the Vochysiaceae. The quantitative distribution of monosaccharides was similar between the species of Qualea and Callisthene, characterized by the predominance of arabinose and mannose, and between the species of Salvertia and Vochysia, which contained higher amounts of arabinose and galactose. Such results are consistent with affinities inferred from floral morphology, wood anatomy and molecular data. Substantial intraspecific variation was observed for some species. UPGMA analysis based on the distribution of the monosaccharides reveals two main clusters, according to the links commented above. The resultant phenogram is not coherent with the current sectional classification of the Vochysiaceae, but the differences in the monosaccharides distribution between the two clusters are strongly supported by ANOVA.
The authors investigated the efficacy of two fluorescein-conjugated lectins (FCLs), concanavalin A (F-ConA) and wheat germ agglutinin (F-WGA), to visualize microorganisms from clinical specimens with documented mycotic and acanthamoebic keratitis. Corneal scrapings from 18 patients with culture-proven keratomycosis and deparaffinized histopathologic specimens from five culture-proven cases of acanthamoebic keratitis were evaluated. The F-ConA provided consistently bright staining of the mycotic structures in each of the corneal scrapings. Both F-ConA and F-WGA stained acanthamoebic trophozoites and cysts in the histopathologic specimens. In both the corneal scrapings and the histopathologic specimens, the microorganisms were easily differentiated from background corneal cells and tissue. These staining patterns correlate well with the results from experimental studies, and with the known cell wall carbohydrate compositions for fungi and acanthamoebae. This study provides further evidence that FCLs (particularly F-ConA) may eventually become effective first-line stains for the visualization of microorganisms in specimens from ocular infections.
Analysis of the susceptibility and the acquisition of tolerance in Enterococus faecalis towards bile salts showed a nearly instantaneous killing effect and yielded evidence for homologous tolerance as well as for cross-protections. Two-dimensional (2-D) electrophoresis revealed 45 proteins which are amplified in response to the bile salt treatment. These include a set of seven proteins, the synthesis of which is increased not only with the bile salts but also with multiple sublethal stresses of various nature. Characterisation of the latter (called general stress proteins) showed that at least five of them are related to resistance to bile salts, heat, ethanol, oxidative and alkaline pH stresses and are probably involved in cross-protection development. On the other hand, random mutagenesis of E. faecalis allowed the isolation of 10 bile salt-sensitive mutants. Their characterisation revealed that the mutation loci corresponded to genes related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition or cell wall synthesis. Further characterisation of one mutant revealed that the insertion within the E. faecalis sagA gene led to morphology changes, to perturbations of cell division and to a decrease of the resistance towards several independent physicochemical stresses.
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Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.
Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of approximately 100 sequential alpha-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.
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To analyse substantial equivalence of genetically modified sugar-beets and maize, in which the glufosinate-tolerant (Pat) gene is inserted, crude nutrients, the amino acid and the fatty acid profiles as well as the composition of the NDF-fraction of maize grains were determined and compared with those of the corresponding non-transgenic cultivars. Due to the genetic manipulation differences in crude nutrient contents including sugar and starch were not detected. The amino acid profile of maize grains was analysed to be the same. Fatty acid profile and composition of cell wall constituents did not show any influences as well. Digestibility of Pat-sugar-beets and maize grains for pigs did not demonstrate meaningful differences as compared to the corresponding non-transgenic cultivars. Digestibility of sugar-beet roots and sugar-beet top silage for ruminants proved to be also in the scope of natural variance. As the digestibility of the macro nutrients remained unaffected, the Pat-gene introduction into both crops did not show an influence on the energetic feeding value. For pigs the ME-content of Pat-sugar-beets was determined to be 14.1 MJ/kg DM versus 13.7 MJ of the non-transgenic cultivars. ME-content of Pat-maize grains was 16.0 MJ/kg DM versus 15.8 MJ for controls. For ruminants the feeding value of Pat-sugar-beets was found to be 8.5 MJ NEL/kg DM or 13.2 MJ ME/kg DM, regardless of whether the Pat-gene was inserted or not. The corresponding energy values of sugar-beet top silage ranged between 5.2 and 5.5 MJ NEL/kg DM or 8.6 and 9.1 MJ ME/kg DM, with differences considered in the biological range.
Strains of a new species, Staphylococcus vitulus, were isolated from food and a variety of mammals. This species was recognized on the basis of the results of an analysis of genomic EcoRI fragments containing portions of the rRNA operons. The patterns of hybridized fragments obtained from strains belonging to the new taxon were sorted into a distinguishable cluster and were distinct from the Staphylococcus lentus and Staphylococcus sciuri patterns. The results of DNA-DNA hybridization reactions demonstrated that strains in this cluster were more closely related to S. lentus and S. sciuri than to other Staphylococcus species and yet were significantly different. While these strains had some of the phenotypic characteristics of the S. sciuri species group, the newly recognized taxon could be distinguished by its very small colonies on P agar, absence of alkaline phosphatase activity, and lack of acid production from L-arabinose, maltose, N-acetylglucosamine, D-mannose, and raffinose. The type strain of the new species is strain DD 756 (= ATCC 51145).
Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (70 degrees C) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. carnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnaticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).
A phenotypic study has been carried out on six moderately halophilic gram-positive nonmotile cocci isolated from ponds of a saltern located in Huelva, Spain. These strains were examined for 150 morphological, physiological, biochemical, and nutritional traits and showed phenotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius). The guanine-plus-cytosine (G + C) content of their DNA ranged between 70 and 72 mol%, values quite similar to those described for N. halobia (71.5 mol%). The 16S rDNA sequence analysis of one representative isolate showed that it is phylogenetically quite close to N. halobia, within the high-G + C-content gram-positive branch. DNA-DNA hybridization experiments showed a high degree of homology (72 to 100%) among the six isolates and the type strain N. halobia ATCC 21727. All data demonstrate quite clearly that the six isolates are members of the species N. halobia. Since this species was described on the basis of a single strain isolated from unrefined solar salt, and its description is not complete (especially in the utilization of different compounds), our study contributes to a better description of the moderate halophile N. halobia.
In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".