Sato H, Kudo S, Ohnishi K, Mizuguchi M, Goto E, Suzuki K
Department of Chemistry, Faculty of Science, Kanazawa University, Japan.
Eur J Biochem. 2001 Apr;268(8):2229-38. doi: 10.1046/j.1432-1327.2001.02098.x.
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.
从恶臭假单胞菌S-1的染色体DNA中克隆了由1266个核苷酸组成的编码水杨酸羟化酶的sal基因并进行了测序[铃木，K.，水口，M.，大西，K.和板垣，E.（1996年）《生物化学与生物物理学学报》1275，154 - 156]。在此，我们描述了sal基因调控区的核苷酸序列以及一个与sal基因反向排列的开放阅读框（salR基因），该基因编码SalR蛋白。此基因产物在转录水平上正向调控sal基因的表达。salR基因由起始于GTG密码子的930个碱基对组成，编码一个309个氨基酸的蛋白质，分子量为34542 Da。其氨基酸序列与恶臭假单胞菌RB1的CatR等LysR家族调控蛋白同源，并且在其N端附近具有螺旋 - 转角 - 螺旋DNA结合基序。确定sal和salR基因的转录起始位点分别位于各自起始密码子上游30个和24个碱基处，且彼此相隔78个核苷酸。在sal和salR基因中可见Shine - Dalgarno序列以及包含 - 10和 - 35序列的推定启动子序列。通过DNA迁移率变动分析证实了salR基因在大肠杆菌细胞中的质粒上的表达。为了过量表达salR基因，将其克隆到pET28a（pSAHR）中，然后转移到大肠杆菌BL21（大肠杆菌BL21/pSAHR）中，并通过诱导剂异丙基硫代 - β - D - 半乳糖苷进行表达。从无细胞提取物中进一步将SalR纯化至同质，产量约为每升培养体积3 mg。测定其分子量为33 kDa，N端氨基酸序列与从salR基因核苷酸序列推导的序列相同。天然SalR也从恶臭假单胞菌S-1中以极低含量纯化至同质。该蛋白质的性质与在大肠杆菌中表达的SalR相似。
