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

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.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

“…Muconate caused an increase in benA transcription (Bar 4) that was not significantly altered by added benzoate (Bar 5). These results are consistent with studies of a chromosomal benA::lacZ transcriptional fusion in different genetic backgrounds (2,5). The level of benA expression achieved with muconate in vitro was similar for CatM-His and BenM-His.…”

“…In mutants lacking BenM, CatM does not activate benABCDE expression to levels sufficient for growth on benzoate as the carbon source. However, CatM may stimulate low-level inducible expression of this operon and may repress basal transcription under some growth conditions (5). As reported here, the transcription of benA in response to two inducers did not require CatM, and the studies of transcriptional synergy centered on BenM.…”

“…However, in mutants lacking BenM, CatM-activated benA expression is insufficient to support growth on benzoate (2, 5). The growth-limiting factor in these strains seems to be the level of CatM-activated benA expression, because several point mutations each allow BenM-independent growth on benzoate by increasing CatM-dependent muconate-activated benA transcription (2,5). Thus, the ability of BenM, but not CatM, to respond synergistically to benzoate in combination with muconate is significant, because it raises benA expression above the threshold needed for growth.…”

Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida

“…The importance of intracellular CCM concentration for the regulation of expression of catabolic operons is also seen in Acinetobacter baylyi ADP1 (Collier et al, 1998;Cosper et al, 2000;Ezezika et al, 2006). At the same time, the accumulation of high levels of CCM is toxic to the cells (Gaines et al, 1996).…”

The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1

“…4). Similarly, in a mutant lacking both BenM and CatM, the expression of benA is approximately fourfold higher than in strains with the regulators when no inducer is present (Cosper et al, 2000). This increased expression results from the ability of CatM or BenM, in the absence of inducers, to bind to a segment of P bA and repress basal benA expression by preventing access to RNA polymerase (Bundy, 2001).…”