Nucleotide sequence analysis of 5'-flanking region of salicylate hydroxylase gene, and identification and purification of a LysR-type regulator, SalR.

Abstract

The sal gene comprised of 1266 nucleotides encoding salicylate hydroxylase was cloned from the chromosomal DNA of Pseudomonas putida S-1 and sequenced [Suzuki, K., Mizuguchi, M., Ohnishi, K. and Itagaki, E. (1996) Biochim. Biophys. Acta 1275, 154-156]. Here, we describe the nucleotide sequences of the regulatory region of the sal gene and an ORF (salR gene) divergently oriented from the sal gene, which encodes the protein SalR. This gene product positively controls sal gene expression at the transcriptional level. The salR gene consists of 930 base pairs starting from a GTG codon and encodes a protein of 309 amino acids with a molecular mass of 34 542 Da. The amino-acid sequence is homologous to LysR-family regulatory proteins such as CatR of P. putida RB1 and has helix-turn-helix DNA binding motif near its N-terminal. Transcription start sites of sal and salR genes were determined to lie 30- and 24-bp upstream of the respective initiation codons and separated from each other by 78 nucleotides. A Shine-Dalgarno sequence and the putative promoter sequences containing -10 and -35 sequences were seen in the sal and salR genes. Expression of the salR gene on a plasmid in Escherichia coli cells was confirmed by DNA mobility shift assay. For the overexpression of the salR gene, it was cloned to pET28a (pSAHR) which was transferred to E. coli BL21 (E. coli BL21/pSAHR), and expressed by an inducer, isopropyl thio-beta-D-galactoside. SalR was further purified to homogeneity from the cell-free extracts in yields of approximately 3 mg.L-1 culture volume. The molecular mass was determined to be 33 kDa and the N-terminal amino-acid sequence was the same as that deduced from the nucleotide sequence of salR gene. Native SalR was also purified to homogeneity from P. putida S-1 with very low contents. The properties of the protein were similar to those of SalR expressed in E. coli.