Chemical composition of cell walls and membranes of yeasts.
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Schizosaccharomyces pombe thermosensitive mutants requiring the presence of an osmotic stabilizer to survive and grow at a nonpermissive temperature were isolated. The mutants were genetically and biochemically characterized. In all of them, the phenotype segregated in Mendelian fashion as a single gene which coded for a recessive character. Fourteen loci were defined by complementation analysis. Studies of cell wall composition showed a reduction in the amount of cell wall beta-glucan in three strains (JCR1, JCR5, and JCR10) when growing at 37 degrees C. Galactomannan was diminished in two others. Strains JCR1 and JCR5, with mutant alleles cwg1-1 and cwg2-1, respectively, were further studied. The cwg1 locus was mapped on the right arm of chromosome III, 18.06 centimorgans (cM) to the left of the ade5 marker; cwg2 was located on the left arm of chromosome I, 34.6 cM away from the aro5 marker. (1-3)beta-D-Glucan synthase activities from cwg1-1 and cwg2-1 mutant strains grown at 37 degrees C were diminished, as measured in vitro, compared with the wild-type strain; however, Km values and activation by GTP were similar to the wild-type values. Mutant synthases behaved like the wild-type enzyme in terms of thermostability. Analyses of round shape, lytic behavior, and low (1-3)beta-D-glucan synthase activity in cultures derived from ascospores of the same tetrad showed cosegregation of all these characters. Detergent dissociation of (1-3)beta-D-glucan synthase into soluble and particulate fractions and subsequent reconstitution demonstrated that the cwg1-1 mutant was affected in the particulate fraction of the enzymatic activity while cwg2-1 was affected in the soluble component. The antifungal agents Papulacandin B and Aculeacin A had similar effects on the enzymatic activities of the wild type and the cwg2-1 mutant strain, whereas the cwg1-1 mutant, when growing at 37 degrees C, had a more inhibitor-resistant (1,3)beta-D-glucan synthase. It is concluded that the cwg1+ and cwg2+ genes are related to (1,3)beta-D-glucan biosynthesis.
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The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.
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A nonlinear electroelastic model for the composite wall of the cochlear outer hair cell is proposed. The cell wall is modeled as a two-layer shell with elastic connections between the layers: an active layer corresponds to the plasma membrane and a passive layer corresponds to a combination of the cytoskeleton and the subsurface cisternae. As a basis of the constitutive relations, a thermodynamic potential for such a composite wall is developed. Expressions for the components of the active force are obtained in terms of the active strains and the elastic properties of the passive and active layers. An application to the electrical stimulation of the cell under the conditions of the microchamber experiment is given. As a result, active strains, active forces, and mechanical energy stored in each of the two layers are presented as functions of the wall (membrane) potential.
Cell wall composition of a stable suspension of proso millet (Panicum miliaceum L. cv Abarr) cells is similar to those of tissues and cell suspensions of other graminaceous species. Extraction of hemicelluloses with step-wise increasing concentrations of alkali yields materials that, like those of embryonal cells of maize coleoptiles, comprise mostly glucuronoarabinoxylan, xyloglucan, and small amounts of (1-3),(1-4)-beta-d-glucan. As in the walls of embryonal cells of the maize coleoptile, 5-arabinosyl and 3-arabinosyl comprise much higher proportions of the total hemicellulosic sugars than in walls of developed or elongated cells. Unlike cells of many dicotyledonous species, millet cells do not elongate or undergo observable differentiation during the stationary phase of culture, and consequently, their wall composition is remarkably consistent throughout the culture cycle. The proso millet cell suspension culture constitutes a reasonable model for study of cell wall biogenesis in embryonal cells of a graminaceous species, but because of marked changes in the composition of hemicelluloses in these species during cell enlargement, additional model systems should be sought.
Red-osier dogwood (Cornus stolonifera Michx, Syn. Cornus sericea), a species relatively well adapted to moderately saline conditions compared with other boreal species, was used to test the effects of NaCl on plant water relations, cell wall elasticity, and cell wall composition of seedlings. Three month-old seedlings were treated hydroponically with 0, 25, and 50 mm NaCl for 21 days. The osmotic potential at full turgor, osmotic potential at turgor loss, pressure potential at full turgor, and relative water content at turgor loss of red-osier dogwood shoot tissue were not significantly affected by the NaCl treatments. Cell wall elasticity of the shoot tissues did not change following NaCl treatments, suggesting that elastic adjustment did not play a role in the adaptation mechanism. Hemicellulose content of the cell wall increased in salt treated seedlings. The primary sugar found in the cell wall hemicellulose fraction was xylose. In the pectin fraction arabinose and galacturonic acid were the main sugars. Sodium chloride stress did not alter the sugar composition of the hemicellulose fraction; however, NaCl did increase the amount of rhamnose in the pectin fraction. The results of this study suggest that at moderate salinity red-osier dogwood does not make any osmotic or elastic adjustments in the shoot tissue, but some changes in the cell wall composition do occur. These changes could contribute to the decrease in growth recorded in red-osier dogwood during NaCl stress.
Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.
In order to study changes in the cell-wall composition of growing poplar cambium during the seasonal cycle, cell walls were isolated from the cambium and newly formed vascular tissues of poplar branches at three times during the year (winter, beginning of spring, end of summer). Polysaccharide material was isolated by sequential extraction of the cell walls and analysed. The principal polysaccharides identified were pectins and xylose- and glucose-containing polysaccharides, possibly xylans and (xylo)glucans. Our results indicate changes in the relative quantities of these polysaccharides during the seasonal cycle.
To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.
Whole bacterial cells of Staphylococcus aureus as well as purified staphylococcal peptidoglycan (PG) have been demonstrated to stimulate human monocytes to release cytokines. Hypothesising that the phenomenon of changes induced by beta-lactam antibiotics in cell-wall composition may alter the immunological properties of the intact cell wall as well as of purified cell-wall components, this study assessed whether cytokine release by human monocytes is altered if cells from strains grown in the presence or absence of sub-minimal inhibitory concentrations of oxacillin are used as stimuli. Whole bacterial cells and isolated PG from these strains, grown in the presence of oxacillin, showed a significantly reduced stimulation of tumour necrosis factor-alpha, interleukin (IL)-1beta and IL-6 release by human monocytes in a concentration-dependent fashion. The serum-induced potentiation of cytokine production by human monocytes in response to PG with modified cross-linking was also reduced. These observations may have particular relevance for staphylococcal infections, in which clinically achievable beta-lactam concentrations do not suppress staphylococcal growth yet may alter and possibly enhance virulence.