Prediction of gene function in methylthioadenosine recycling from regulatory signals

Abstract

The S-box transcription termination control system, first identified in Bacillus subtilis, is used for regulation of gene expression in response to methionine availability. The presence of the S-box motif provided the first indication that the ykrTS and ykrWXYZ genes could play a role in recycling of 5'-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine. In this study we demonstrate a role for the ykrTS and ykrWXYZ gene products in this pathway.

FIG. 1.

Methionine biosynthesis pathways in B. subtilis. Genes that are preceded by S-box regulatory elements are shown with a boxed “S.” The “y” designation in gene names indicates genes whose function has not been experimentally established; possible assignments to enzymatic steps demonstrated in K. pneumoniae (6, 19) are shown but have not yet been tested (8). The function of the ykrS gene product is not known. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SRH, S-ribosylhomocysteine; MT, methylthio; KMTB, 2-keto-4-methylthiobutyrate; THF, tetrahydrofolate.

FIG.2.

Growth of ykrTS and ykrWXYZ mutants. ykrS, ykrW, and yitJ mutants were generated by insertional inactivation by Campbell recombination of a derivative of plasmid pBEST 501 (10) containing an internal fragment of the target gene into the chromosome of strain BR151MA (lys-3 trpC2). The ykrT mutant was generated by homologous recombination, using a plasmid in which the ykrT coding region was deleted and the ykrS coding region was placed immediately downstream of the ykrT leader region. Strain constructions were verified by PCR and DNA sequencing. Cultures were grown overnight in Spizizen minimal medium (1) containing methionine (0.34 mM), and cells were collected by centrifugation and resuspended in medium containing sulfate alone (open circles), methionine (filled circles), MTA (Sigma) at 0.4 mM (squares), or MTR at 0.4 mM (triangles). MTR was prepared by acid hydrolysis of MTA as described by Schlenk et al. (13). (A) BR151-ZKO (metB10); (B) BR151-YkrTKO (metB10 ΔykrT); (C) BR151-YkrSKO (metB10 ykrS::neo); (D) BR151-YkrWKO (metB10 ykrW::neo); (E) BR151MA-YitJKO (yitJ::neo).