The first major goal of this project is to complete the genetic analysis of the physiological function of peptidases in Salmonella typhimurium. To do this we will: a) isolate mutations affecting all of the remaining major peptidases of the cell; b) use these mutants to define the role of the individual enzymes in the protein turnover pathway and to study the effect of their loss on cell growth and viability; and, c) characterize the regulation of these peptidase genes. Studies of peptidase regulation will require analysis of DNA sequence changes and studies of the role of individual peptidases on the degradation pathway will require purified enzymes. We therefore propose to isolate cloned pep genes for use in obtaining DNA for the analysis of sequence changes in regulatory mutants and for use in obtaining enzyme overproducers. The second major goal of the project is to begin genetic analysis of endoprotease function in Salmonella. We plan to isolate and genetically characterize mutations affecting the major endoproteases of the cell. We intend to use peptide substrates in screening techniques similar to those already used for the isolation of peptidase mutants. Characterization of the physiological consequences of these mutations should show which enzymes carry out which steps in the protein degradation pathway and in other proteolytic processes. The sequence of steps by which a protein molecule is degraded constitutes a major metabolic pathway occurring in all cells. Little is known about the roles of individual enzymes in this pathway in any organism. The use of a genetic approach to the study of this problem seems especially appropriate given the large number of peptidases and proteases and the broad overlap in their substrate specificities. Well developed techniques allow the construction of organisms lacking specific enzyme activities or combinations of activities. The usefulness of bacteria for the production of peptide and protein gene products of cloned DNA from non-bacterial sources probably depends at least in part on the construction of strains that will not proteolytically destroy these products. An understanding of the specificities and regulation of cellular proteases and peptidases may be crucial for the construction of such strains.
| Solbiati, J; Chapman-Smith, A; Miller, J L et al. (1999) Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal. J Mol Biol 290:607-14 |
