Abstract

S-adenosylmethionine(SAM) is synthesized from methionine by SAM synthetase. In E. coli this enzyme is encoded by metK . Since metK is an essential gene and SAM cannot cross bacterial cell membranes, it is difficult to study the role of SAM directly in vivo. We introduced the SAM transporter gene from Rickettsia (kindly provided by Dr. D. O. Wood) to study the effect of exogenous supply of SAM on expression of genes of methionine biosynthesis in E. coli strains carrying a deletion of metK . Such strains could not make their own SAM but could transport SAM provided in the medium. The effect of growth with SAM, and SAM starvation on expression of the genes encoding most of enzymes in methionine biosynthesis, metA, metB, metC, metE, metF, metK, metR, was studied by means of reporter gene constructs using their promoters fused to lacZ . The Beta-Galactosidase coded by lacZ will reflect the met genes' expression in the SAM including medium or non-SAM medium. By using the above system, the effect of SAM on regulating met genes was studied. The result showed that in rich medium depleting the SAM supply of the E metK strain dramatically induced metB and metR (both 19.2 fold). The other genes were little affected. During growth in minimal medium with methionine and glucose, depleting the SAM pool had a much greater effect, inducing all the genes tested 10 fold to 100 fold. Moreover, there is no significant increase for all met genes in metJ deletion strain. These results support the in vitro model of MetJ-SAM repressor complex binding met gene promoters to block their transcription. Furthermore, result of studying a metJ deficient mutant MNR6 implies that the truncated MetJ in MNR6 retains a considerable function. All these studies give one step further to understand the mechanism of regulating met gene expression by MetJ-SAM complex in vivo.