Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene

Abstract

Regulation of the SalmoneUla typhimurium metJ gene was examined by measuring j8-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metj-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pjl and PJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters Pjl and PJ2 individualHy. Promoter Pjl was negatively regulated by the metJ gene product and by methionine. Although PJ2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJl.In both Salmonella typhimurium and Escherichia coli the metJ gene codes for a protein that is involved in the regulation of the methionine pathway (5,13,17). This protein interacts with S-adenosylmethionine to form an active complex that presumably functions as a classical repressor (13,17). The metJ gene of S. typhimurium has been cloned and its nucleotide sequence has been determined (18,19). To facilitate studies on regulation of the metJ gene, we have fused the upstream control sequences for this gene to the E. coli lacZ gene. Deletion derivatives of this fused gene have been constructed to test a model proposing that the metJ gene is transcribed from two distinct interacting promoters (19). MATERIALS AND METHODSBacterial strains, phage, and plasmids. Descriptions and origins of bacterial strains are given in Table 1. All bacterial strains are derivatives of E. coli K-12. Plasmid pGS107 was described previously (18). Plasmid pMC1403 (2) was from M. Casadaban. Phage Xgt2 (10) was from R. Davis. Other plasmids and phage were isolated during this investigation.Media. Luria broth and glucose minimal media have been described (16). Supplements were added at the following concentrations: phenylalanine, 50 ,ug/ml; vitamin B1, 1 pLg/ml; D-methionine, 150 ,ug/ml; L-methionine, 50 ,ug/ml; ampicillin, 100 ,ug/ml; 5-bromo-4-chloro-3-indolyl-f3-Dgalactoside (X-gal), 40 ,ug/ml. DNA manipulations. The general procedures used for restriction enzyme cleavage, ligation, plasmid and phage DNA isolation, isolation of DNA fragments from polyacrylamide gels, and transformation have been described (6). DNA sequence analysis was done by the method of Maxam and Gilbert (7).Construction of plasmids pBlac and pJlac. A 480-base pair (bp) RsaI DNA fragment isolated from plasmid pGS107 that carries the promoters and the coding sequences for the amino-terminal ends of the S. typhimurium metB and metJ genes was ligated in both orientations into the SmaI site of plasmid pMC1403 (Fig. 1). The RsaI cleavage sites occur between codons in the metB and metJ genes and therefore maintain the reading frame of the lacZ gene in plasmid pMC1403. The ligation products were then used to transform strain GS563, the cells were plated on Luria broth-ampicillin * Corresponding author.plates containing X-gal, and blue colonies were...

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

“…metJ is autoregulated, as shown by the repressive effect of excess levels of the aporepressor on metJ expression with or without added SAM (Shoeman et al, 1985;Urbanowski & Stauffer, 1986). SAM enhances this autoregulatory activity, so the removal of SAM by SAMase may simply decrease the effectiveness of this regulation, requiring higher concentrations of MetJ to regulate its own gene.…”

“…They were identified by screening for resistance to 50 mg kanamycin ml 21 on YT agar plates after P1 vir transduction and subsequent demonstration of elevated MetC activity using the assay described below. E. coli BW545, BWmJ (metJ : : Tn5) and BWmK (metK : : Tn5) lysogenic for bacteriophage lgt2 constructs containing Salmonella typhimurium met gene promoter : : lacZ fusions were assayed for metB (lBlac; Urbanowski & Stauffer, 1986), metE (lElac; Plamann et al, 1988), metF (lFlac; , metH (lHlac; Urbanowski & Stauffer, 1989b), metJ (lJlac; Urbanowski & Stauffer, 1986) and metR (lRlac; expression. In all cases, cells used to inoculate liquid cultures came from colonies raised overnight on agar plates inoculated with either freshly transformed cells or transformants stored in 15 % (v/v) glycerol at 270 uC and streaked for purification.…”

Role of the sRNA GcvB in regulation of cycA in Escherichia coli

“…The intermediate plasmid was designated pcycA : : lacZ. A 5788 bp EcoRI-MfeI fragment from pcycA : : lacZ carrying the cycA : : lacZYA fusion was then ligated into the EcoRI site of phage lgt2 (Panasenko et al, 1977), and the phage used to lysogenize E. coli host strains as described previously (Urbanowski & Stauffer, 1986). Each lysogen was tested to ensure that it carried a single-copy of the l chromosome by infection with lcI90c17 (Shimada et al, 1972).…”

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

“…Appropriate strains were lysogenized with XElac or XElacl fusion phage by the procedure described previously (19). After purification, the lysogens were tested for a single copy of the X phage by infection with phage X c190 c17 (17).…”