Cosper N J, Collier L S, Clark T J, Scott R A, Neidle E L
Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
J Bacteriol. 2000 Dec;182(24):7044-52. doi: 10.1128/JB.182.24.7044-7052.2000.
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.
筛选出不动杆菌属ADP1菌株的突变体，使其在没有BenM转录激活因子的情况下利用苯甲酸盐生长。在野生型中，BenM对苯甲酸盐和顺，顺-粘康酸作出反应，以激活benABCDE操纵子的表达，该操纵子参与苯甲酸盐的分解代谢。这个操纵子编码将苯甲酸盐转化为儿茶酚的酶，儿茶酚是一种随后由cat基因编码的酶降解的化合物。在本报告中，发现四个自发突变体携带catB突变，这些突变使得它们能够在没有BenM的情况下利用苯甲酸盐生长。catB编码粘康酸环异构酶，这是苯甲酸盐分解代谢所需的一种酶。其底物顺，顺-粘康酸是由catA编码的儿茶酚1,2-双加氧酶从儿茶酚酶促产生的。从野生型和catB突变体中纯化出均一的粘康酸环异构酶。每种纯化的酶都有活性，尽管野生型和变异型粘康酸环异构酶的催化特性存在差异。构建了具有染色体benA::lacZ转录融合的菌株，并用于研究catB突变如何影响在苯甲酸盐上的生长。所有的catB突变都增加了缺乏BenM的菌株中顺，顺-粘康酸激活的ben基因表达。提出了一个模型，其中catB突变在苯甲酸盐生长过程中降低了粘康酸环异构酶的活性，从而增加了细胞内顺，顺-粘康酸的浓度。反过来，这可能使CatM（一种在序列和功能上与BenM相似的激活因子）激活ben基因转录。CatM通常对顺，顺-粘康酸作出反应以激活cat基因表达。与该模型一致，相对于野生型，在以苯甲酸盐生长的catB突变体细胞提取物中的粘康酸环异构酶比活性较低。此外，突变体的儿茶酚1,2-双加氧酶活性升高，这可能是由于CatM对细胞内顺，顺-粘康酸水平的改变作出反应并增加了catA表达所致。总的来说，这些结果支持了代谢物浓度在通过复杂的转录调控回路控制苯甲酸盐降解中的重要作用。
