Cell wall composition and ultrastructure of the extremely halophilic coccus, sarcina marina
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Cells of S. marina (N.C.M.B, 778) were disrupted using a Hughes press and a purified cell wall fraction obtained using a previously reported method for halococcal wall isolation. This procedure was monitored by examination of thin sections in the electron microscope and the final wall preparation was seen to be relatively free of cytoplasmic and membranous contaminants. However, treatment of the wall fraction with crude trypsin did appear to remove particulate surface components. The total ninhydrin-positive components detectable accounted, for only about 14% of the cell wall dry weight. The major amino acids present were glycine, alanine, glutamic acid and aspartic acid although very small amounts of others were detected. The amino sugar components included glucosamine and galactosamine although these only accounted for some 60% of the total amino sugars. The remainder was probably made up of one or more of four unidentified, acid-labile components detected on amino acid analysis and by paper chromatography. This is in accord with the finding of unusual, labile amino sugars in the cell walls of other halococcal species. Approximately 37% of the cell wall dry weight was made up of the neutral sugars, glucose, galactose and mannose which were present in eguimolar amounts. In addition, the wall was found to contain a negligible lipid (0.1% dry weight) and a high ash (9.2% dry weight) content. The poor recovery of organic material after analysis is almost certainly due to the lability of some of the more unusual (and in this work unidentified) components. Attempts to selectively solubilise the wall material with a view to identifying discrete polymers met with some success. In Particular, treatment with trichloroacetic acid (TCA) at 35° extracted all of the glactosamine from the wall (in addition to other components) but none of the unknown component, X1. Further treatment with TCA at 60 extracted all of another unknown component, X1. These results suggest that some degree of resolution of different polymers constituting the wall may be possible and may have been achieved here. Treatment of S. marina with the antibiotics, D-cycloserine, novobiocin, bacitracin, penicillin G and vancomycin, known to affect cell wall biosynthesis in other bacteria, was carried out. Possible effects of the antibiotics were monitored by electron microscopy and turbidimetric estimation of bacterial growth. Only novobiocin and bacitracin had any effect on growth but this was marked; in both cases growth was prevented by addition of the antibiotic. The other three antibiotics all lost their antibiotic activity (against appropriate indicator organisms) when incubated over a period of a few hours in Dundas medium. It is suggested that this may be a significant consideration when explaining the antibiotic insensitivity of microorganisms, such as S. marina whose doubling times are of the same order of magnitude as that necessary for antibiotic inactivation. Thin sections of control and antibiotic-treated cells showed interesting ultrastructural features comparable with those seen in more conventional halophilic cocci. Some minor ultrastructural changes were seen in some of the anti-biotic-treated cells, the most notable being extensive plasmolysis in the case of novobiocin. However, none of the antibiotics tested appeared to cause cell lysis or osmotic fragility which may preclude their use as agents for the non-destructive removal of the cell wall.
Thesis, PhD Doctor of Philosophy
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