Cowles C E, Nichols N N, Harwood C S
Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242, USA.
J Bacteriol. 2000 Nov;182(22):6339-46. doi: 10.1128/JB.182.22.6339-6346.2000.
Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded meta pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of P. putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease. Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A benR mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The benABC genes likely encode benzoate dioxygenase, and benD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase. benK and benF were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene, benE, is not known. benR activated expression of a benA-lacZ reporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benR was required for benzoate-mediated repression of pcaK-lacZ fusion expression. The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.
恶臭假单胞菌利用两种由染色体编码且其表达受苯甲酸诱导的酶将苯甲酸转化为儿茶酚。苯甲酸还与调节因子XylS结合，以诱导TOL（甲苯降解）质粒编码的间位途径操纵子的表达，该操纵子用于降解苯甲酸和甲基苯甲酸。最后，苯甲酸通过阻止编码4-羟基苯甲酸通透酶的基因pcaK的转录，抑制恶臭假单胞菌转运4-羟基苯甲酸（4-HBA）的能力。在此，我们鉴定出一个基因benR，它是苯甲酸、甲基苯甲酸和4-HBA降解基因的调节因子。通过随机转座子诱变分离得到的benR突变体无法在苯甲酸上生长。BenR推导的氨基酸序列与调节因子AraC家族成员XylS的序列具有高度相似性（62% 同一性）。基于其推导的氨基酸序列，推断与benR相邻的另外七个基因参与苯甲酸降解。benABC基因可能编码苯甲酸双加氧酶，benD可能编码2-羟基-1,2-二羟基苯甲酸脱氢酶。benK和benF分别被赋予苯甲酸通透酶和孔蛋白的功能。最后一个基因benE的可能功能尚不清楚。benR响应苯甲酸激活benA - lacZ报告基因融合体的表达。它还激活插入大肠杆菌染色体中的间位裂解操纵子启动子 - lacZ融合体的表达。第三，benR是苯甲酸介导的pcaK - lacZ融合体表达抑制所必需的。benA启动子区域包含一个直接重复序列，该序列与先前为间位裂解操纵子启动子定义的XylS结合位点相匹配。BenR可能结合染色体苯甲酸降解基因和质粒编码的甲基苯甲酸降解基因的启动子区域，以响应苯甲酸激活基因表达。BenR在抑制4-HBA摄取中的作用可能是间接的。
