Identification of a mutation in the Bacillus subtilis S-adenosylmethionine synthetase gene that results in derepression of S-box gene expression

Abstract

Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5' region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

FIG. 1.

The S-box regulatory system. In the absence of SAM, the antiterminator structure forms (AT, red-blue), preventing formation of the competing terminator helix (T, blue) and allowing transcription of the downstream coding sequence. Binding of SAM to the helix 1 to 4 region results in stabilization of helix 1, which serves as an anti-antiterminator (AAT, black-red) by sequestering sequences necessary for formation of the antiterminator, thereby allowing formation of the terminator helix and transcription termination. SAM binding also promotes a tertiary interaction (dashed line) between residues (green) in the terminal loop of helix 2 and the unpaired region between helices 3 and 4. SAM (*) is generated from methionine and ATP by MetK (SAM synthetase).

FIG. 2.

Sequence comparison of bacterial MetK homologs. The region of B. subtilis MetK surrounding residue A83 is aligned with the analogous MetK regions from other bacterial species. Sequences were obtained by searching the NCBI database with signature sequences for SAM synthetases (6) by using the tblastn program (1). A83 in B. subtilis MetK and the analogous residues from the other bacterial MetKs are boxed. Residues that differ from the B. subtilis sequence are shown in boldface. Bsu, B. subtilis; Ban, Bacillus anthracis; Sau, Staphylococcus aureus; Lpl, Lactobacillus plantarum; Spn, Streptococcus pneumoniae; Sty, Salmonella enterica serovar Typhimurium; Bma, Burkholderia mallei; Pae, Pseudomonas aeruginosa; Bpe, Bordetella pertussis; Eco, E. coli. (+), gram-positive species; (−), gram-negative species. Amino acid identities to B. subtilis MetK were as follows: 86%, B. anthracis; 77%, S. aureus; 68%, L. plantarum; 68%, S. pneumoniae; 63%, S. enterica serovar Typhimurium; 59%, B. mallei; 59%, P. aeruginosa; 58%, B. pertussis; 57%, E. coli.

FIG. 3.

Measurement of SAM pools in cell extracts of metK⁺ and metK10 strains by SAM-dependent transcription termination. Cell extracts were generated from strains BR151MA and BR151MA-SBD1 by formic acid extraction of cells grown in minimal medium in the presence of methionine. Extracts were neutralized by the addition of 1 N KOH, and samples (standardized by cell numbers used in preparation of the extracts) were added to a B. subtilis RNAP in vitro transcription reaction mixture containing a P_gly-yitJ DNA template. The percent termination was calculated as the fraction of the terminated RNA product relative to the total of the terminated and readthrough products. Reproducibility was ±5%. Measurement of termination efficiency in response to addition of various amounts of SAM resulted in generation of a standard curve in which 50% termination was observed at 0.15 μM SAM. The amount of cell extract required to confer 50% termination was compared to the SAM standard curve. An average internal cell volume of 0.535 ± 0.13 μl A₆₀₀⁻¹ (35) was used to calculate the intracellular SAM concentrations (250 μM for BR151MA and 59 μM for BR151MA-SBD1) from the cell equivalents and A₆₀₀ of cell cultures used in preparation of the extracts.

FIG. 4.

Location of S80 in the crystal structure of the E. coli SAM synthetase ternary complex. The crystal structure of E. coli SAM synthetase (15) with the ATP analog AMPPNP and methionine in each active site is shown. The image was created using the Cn3D v4.1 three-dimensional structure viewer (NCBI), using coordinates from the work of Komoto et al. (15). The tetrameric enzyme is a dimer of dimers, with two active sites located between the two subunits in each dimer. The location of S80 (in tan) in each subunit is indicated by red arrows. S80 is the only residue that participates in hydrogen bond formation between the dimers of E. coli SAM synthetase.