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Digitized by Kansas State University Libraries

Digitized by Kansas State University Libraries

Call number: LD2668 .T4 1963 M45 / Master of Science

Call number: LD2668 .T4 1963 T24 / Master of Science

Call number: LD2668 .T4 1966 H749 / Master of Science

Call number: LD2668 .T4 1951 G37 / Master of Science

The role of calcium and the existence of calmodulin or other calcium-binding proteins in E. coli has received very little attention previously. In this study, it is demonstrated that growth and cell division of E. coli are very sensitive to a group of calmodulin antagonists. Mutants resistant to calmodulin inhibitors have been isolated. The fee mutants are resistant to 48/80, and are temperature sensitive for growth, involving some purtubation of the cell cycle. wseA mutants are resistant to at least two calmodulin antagonists, 48/80 and W-7, and produced a population of filamentous cells. Intensive studies with the feeBl mutant demonstrated that the mutation is located in the acceptor stem of tRNAleu3 which recognises the rare CUA codon. Other cloning studies have also shown that in all probability feeA is also a leucine tRNA gene, leul, which recognises the abundant codon, CUG. The specific base change in the feeAl mutant, however, remains to be identified. The feeBl mutant was shown to be impaired in the synthesis of B-galactosidase (which has 6 CUA codons in its mRNA), at both the permissive and non-permissive temperatures. It is proposed that the synthesis of other polypeptides requiring the translation of several CUA codons is severely reduced in feeB mutants at the restrictive temperature, and is reduced to a suboptimal level for function at the permissive temperature. It is further proposed that if such a polypeptide(s) regulates the level of a specific calcium-binding protein which is inhibited by 48/80, or in some other way affects the level of free intracellular Ca2+, this could lead to the observed resistance to the drug. In the case of feeA, it is difficult to explain the relationship between the mutation of an abundant tRNA and the observed drug resistance. Possible explanations for the action of a mutant tRNALeu1, including an effect upon the level or function of the rare tRNALeu3 are described in Chapter VIII.

The broad aim of this project was to investigate the effects of introducing the Histidine permease of S.typhimurium into M.methylotrophus, a Gram negative bacterium with no known protein transport mechanism. This should not only contribute to the elucidation of the mechanism by which these periplasmic permeases operate, but also help in the understanding of M.methylotrophus an organism of commercial importance whose biochemistry is not well characterised. Although the entire operon encoding this permease had recently been sequenced prior to the commencement of this study, the precise identity of the components had not been established; furthermore, since each component is produced in very small amounts, simple identification by electrophoretic techniques was not possible. My initial aim, therefore was to introduce the structural genes of the operon under the control of a strong, but inducible promoter, inorder that each gene product could be identified and the location of the protein within the cell established. The His P protein was identfied in this manner, however discrepencies in overproduction prevented the precise location of this polypeptide within the cell from being established. Induced expression of the hisQ gene also identified an overproduced polypeptide of 30,000 molecular weight, suggesting this to be the His Q protein, however I was unable to establish a precise identification. The construction if these new plasmid vectors assisted in the second stage of this study; the introduction of the histidine permease into M.methylotrophus. Furthermore, with three out of four protein components formally identified in E.coli these could then be confirmed in M.methylotrophus. The results obtained clearly show, not only that at least His J was synthesised, but also apparently processed and localised correctly into the periplasm of M.methylotrophus. Furthermore, the permease system was found to be functional in M.methylotrophus demonstrating active histidine uptake into the cell.

No description available.