Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2

Abstract

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR(His). TsaR(His) was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR(His) to be a monomer in solution. Overexpressed TsaR(His) bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K(D)) for the whole promoter region was about 0.9 micro M, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.

FIG. 1.

Degradation of TSA and TCA to amphibolic intermediates by C. testosteroni T-2 and the four regulons involved (R1 to R4) (9, 29, 36). Reactions of gene products that are encoded by chromosomal genes are in insets. Regulatory units R1 (pTSA) and R3 (pT2L) are plasmid encoded (; Ruff, unpublished). The tsa regulon is sketched schematically, with the directions of transcription indicated by shaded triangles. TsaMB, p-toluenesulfonate methylmonooxygenase (oxygenase M, reductase B); TsaC, p-sulfobenzylalcohol dehydrogenase; TsaD, p-sulfobenzaldehyde dehydrogenase; PszA(C), p-sulfobenzoate-3,4-dioxygenase.

FIG. 2.

Expression and purification of TsaR^His from E. coli strain M10 monitored by SDS-PAGE (A) and Western blot analysis (B). E. coli M10 was grown in the absence and presence of IPTG, the inducer of expression of tsaR^his. Soluble proteins from disrupted cells were compared; TsaR^His from induced cells was purified, and the His tag was identified immunologically. Lanes 1 and 8, calibration proteins with the molecular mass values given; lane 2, extract of noninduced cells; lane 3, extract of induced cells; lane 4, supernatant fluid after addition of nickel-agarose; lane 5, supernatant fluid from wash 1 of the purification protocol; lane 6, supernatant fluid from wash 2 of the purification protocol; lane 7, purified TsaR^His.

FIG. 3.

Band shift assays with TsaR^His and specific fragments of DNA from C. testosteroni T-2. (A) tsaM^cod, coding region of tsaMB (Table 3) used as a negative control for nonspecific DNA binding; (B) tsa^prom, promoter region between tsaR and tsaM (Table 3); pszA^prom, promoter of pszA (Table 3) used as a control for promoter specificity of DNA binding of TsaR^His.

FIG. 4.

The effect of TSA concentration on the band shift of tsa^prom caused by TsaR^His.

FIG. 5.

Sequence and structure near the tsa promoter region. The nucleotide positions are those of U32622 (13). Data from genes tsaR (both strands) and tsaM are in uppercase characters, with the start codons in bold characters; amino acids are in uppercase italic characters. The putative ribosomal binding site (∗¹), transcriptional start (∗²), Pribnow box (∗³), and −35 (∗⁴) regions are in bold characters and are underlined for tsaR and shaded grey for tsaM. Subfragments 1 to 5 (Table 3) are indicated with pairs of arrows. The dashed line indicates putative DNA-binding sites for TsaR^His in the presence of TSA (I and III), and the dotted line indicates the putative binding site for TsaR^His in absence of TSA (IV); the binding region II is also shown with a dotted line with terminal arrows. An overview depicting subfragments 1 to 5 with the binding sites and the requirement of TSA is sketched below the sequence.