CONSTRUCTION OF A NEW SYSTEM FOR SEPARATE EXPRESSION OF MUTAGENESIS PROTEINS - THE ABILITIES TO PROMOTE UV MUTAGENESIS AND INTERCHANGEABILITY OF MUCA', MUCB, SAMA' AND SAMB PROTEINS IN SALMONELLA-TYPHIMURIUM

The two distinct mucAB and samAB operons originally isolated from the plasmids of Salmonella typhimurium encode proteins engaged in induced mutagenesis. They represent two extreme cases among these far characte rized members of the enterobacterial umuDC family in respect to both t he strength and the specificity of their effect. It is suggested that the MucA and SamA proteins are post-translationally processed to MucA' and SamA', respectively, which lack the N-terminal 25 amino acids and are the active species in mutagenesis. For the purpose of characteriz ing the individual activities of these proteins, we developed a new sy stem for their SOS-independent separate and controllable expression in enterobacteria. Besides the matured forms of MucA', SamA' as well as MucB and SamB proteins we also expressed hybrid HisTag-MucA' and HisTa g-SamA' proteins in which a synthetic 24 amino acid HisTag region repl aces the natural 25 amino acid N-terminal leader present in the MucA a nd SamA precursors. In this study, we analyzed the effect of the mutag enesis proteins on the UV mutability of S. typhimurium YG5144. None of the proteins, if expressed alone, promoted UV mutagenesis. Different combinations of the proteins promoted mutagenesis to different extents in the order MucA' + MucB > SamA' + SamB greater than or equal to His Tag-MucA' + MucB greater than or equal to SamA' + MucB > MucA' + SamB > HisTag-SamA' + SamB. The mutagenesis enhancing potential of the comb inations with MucB protein decreased as the expression of the proteins increased while the mutagenesis enhancing potential of the combinatio ns with SamB protein increased together with the increase in the expre ssion. The artificially expressed MucA' + MucB proteins were as active as their MucAB counterparts expressed from the plasmid pKM101 in prom oting UV mutagenesis, but they were remarkably more efficient than the ir pKM101-born counterparts in promoting spontaneous mutagenesis. We c onclude that the MucA'B and SamA'B proteins are partly interchangeable and the functionality of the resulting A' + B complex is largely depe ndent on the appropriate B-protein.