Mutations in catB, the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distal ben Genes and Contribute to a Complex Regulatory Circuit in Acinetobacter sp. Strain ADP1

Mutants of the bacterium Acinetobacter sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate and cis,cis-muconate to activate expression of the benABCDE operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by cat gene-encoded enzymes. In this report, four spontaneous mutants were found to carry catB mutations that enabled BenM-independent growth on benzoate. catB encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate, cis,cis-muconate, is enzymatically produced from catechol by the catA-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the catB mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomal benA::lacZ transcriptional fusion were constructed and used to investigate how catB mutations affect growth on benzoate. All of the catB mutations increased cis,cis-muconate-activated ben gene expression in strains lacking BenM. A model is presented in which the catB mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular cis, cis-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate ben gene transcription. CatM normally responds to cis,cis-muconate to activate cat gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown catB mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels of cis,cis-muconate and increasing catA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.

本研究筛选得到可在不含BenM转录激活因子（BenM transcriptional activator）的条件下以苯甲酸为唯一碳源生长的不动杆菌属菌株ADP1（Acinetobacter sp. strain ADP1）突变体。野生型菌株中，BenM可响应苯甲酸与顺,顺-粘康酸（cis,cis-muconate），激活参与苯甲酸分解代谢的benABCDE操纵子（benABCDE operon）的表达，该操纵子编码的酶可将苯甲酸转化为儿茶酚，后续儿茶酚可由cat基因编码的酶进一步降解。本研究中共获得4株自发突变体，均携带catB基因突变（catB mutations），该突变可使菌株在缺失BenM的情况下仍能以苯甲酸为碳源生长。catB基因编码粘康酸环异构酶（muconate cycloisomerase），这是苯甲酸分解代谢过程中的必需酶类，其底物顺,顺-粘康酸由catA基因编码的儿茶酚1,2-双加氧酶（catechol 1,2-dioxygenase）催化儿茶酚生成。研究人员将野生型菌株与catB突变体中的粘康酸环异构酶纯化至均一状态，纯化所得酶均具有催化活性，但野生型与突变型粘康酸环异构酶的催化特性存在差异。研究人员构建了携带染色体benA::lacZ转录融合（chromosomal benA::lacZ transcriptional fusion）的菌株，以此探究catB突变如何影响菌株在苯甲酸培养基上的生长，所有catB突变均可在缺失BenM的菌株中增强顺,顺-粘康酸介导的ben基因表达。本研究提出如下调控模型：catB突变会降低菌株在苯甲酸培养基中生长时的粘康酸环异构酶活性，进而提升细胞内顺,顺-粘康酸的浓度，这一变化可使序列与功能均与BenM相似的激活因子CatM激活ben基因的转录。CatM通常可响应顺,顺-粘康酸以激活cat基因的表达，与该模型一致的是，以苯甲酸为碳源培养的catB突变体细胞提取物中，粘康酸环异构酶的比活性显著低于野生型菌株。此外，突变体的儿茶酚1,2-双加氧酶活性有所升高，这可能是由于CatM响应了细胞内顺,顺-粘康酸水平的改变并上调了catA基因的表达。综上，上述实验结果证实了代谢物浓度在通过复杂转录调控回路控制苯甲酸降解过程中的关键作用。