ABSTRACT
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 andcis,cis-muconate to activate expression of thebenABCDE 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 carrycatB 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 chromosomalbenA::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-activatedben 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 ofcis,cis-muconate and increasingcatA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.
UDP-Glucose Dehydrogenases: Identification, Expression, and Function Analyses in Upland Cotton (Gossypium hirsutum)
