Studies of the histidine permease of salmonella typhimurium in escherichia coli and methylophilus methylotrophus.

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.