Biochemical genetics of bacterial sporulation. V. Fine structure analysis of two sporulation mutants of B. subtilis derepressed for late functions.
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Biomedical subjects
Publications and source records attributed to J C Sousa.
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The ultrastructure of the cytoplasmic membrane and cell wall of two strains of Escherichia coli, Proteus morganii, P. vulgaris, Acinetobacter anitratum, Moraxella lacunata, Erwinia amylovora, Acinetobacter sp., and of a plant pathogen, unclassified gram-negative, fixed by the Ryter-Kellenberger procedure, was found to be significantly affected by the use or omission of the uranyl postfixation included in that procedure, and by the presence or absence of calcium in the OsO(4) fixative. The omission of the uranyl treatment results in a less clear profile of both the outer membrane of the cell wall and of the cytoplasmic membrane. The observation of these two membranes is further limited when both uranyl and calcium are omitted. The R-layer and the material covering the surface of the cell wall appear more distinct when the uranyl postfixation is not used. Evidence is given suggesting that the influence of uranyl and calcium ions on the appearance of the outer and cytoplasmic membranes would be primarily due to their action as fixatives, whereas the influence of uranyl on the appearance of the R-layer would be due to a direct action on the peptidoglycan component of this layer. When uranyl acetate is used as a section stain after the embedding in plastic, it improves the observation of the R-layer. In this case, a well contrasted R-layer is consistently observed in all strains studied, provided that the postfixation has been omitted. The frequent difficulty in clearly observing the R-layer in many published micrographs probably results from the common use of uranyl postfixation.
The ultrastructural alterations induced in vegetative, exponentially growing Bacillus cereus cells suspended in broth, by heating at 65 C for 2 and 15 min and 100 C for 5 min, were studied by electron microscopy of thin sections. The following alterations were observed: (i) change in the triple-layered profile of the membranes from the normal asymmetric to a symmetric geometry, appearance of fractures in the membranes, occurrence of prominent myelin-like systems of concentric membranes; (ii) disappearance of the ribosomes in most cells heated at 65 C and in all cells heated at 100 C; (iii) cytoplasmic precipitation resulting in the appearance of dense blocks of Pronase-sensitive material. The cell wall appears unaffected in most preparations. No signs of deoxyribonucleic acid damage are observed. These ultrastructural data are discussed in relation to the alterations in the chemistry and physiology of heated bacteria described in the literature.
Mutations affecting quantitatively the production of the sporulation-associated extracellular alkaline protease were isolated and characterized. They fall into at least five genes, three of which, ScoA, B and C, were mapped in the argC-metC region. The pleiotropic effects of these mutations concern several or all of the following: rate and timing of protease production, synthesis of alkaline phosphatase, time-course of spore formation. Electron microscopic evidence indicates delayed switch from one morphological stage to another. The nature of the Sco mutations and the genetic regulation of sporulation are discussed.
Asymptomatic dogs from a Kala-Azar endemic region were screened for infection status by parasitological, immunological and molecular techniques. Bone marrow was examined for the presence of parasites by NNN culture and by using the Lmet 2 DNA probe. All the samples were negative in culture but 24 of 41 were positive as determined by the probe. Cellular and humoral immunity were detected by T cell proliferation assays and IFAT respectively. Specific cellular and humoral immunity were found in 20 and 26 dogs respectively out of a total of 41 dogs examined. The vast majority of dogs with Leishmania-specific antibodies were found to be parasitologically positive using the DNA probe while almost half those that had demonstrable cellular immunity were apparently parasite free. The observation that dogs can develop cellular immunity following natural infection clearly indicates that there is a spectrum of canine leishmaniasis similar to that observed in the human disease. The prevalence of dog leishmanial infection must also be higher than was presumed.