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.
Cosper
N J
NJ
Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
Collier
L S
LS
Clark
T J
TJ
Scott
R A
RA
Neidle
E L
EL
D000150:Acinetobacter / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins; D001565:Benzoates / Q000378:metabolism*; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C112178:BenM protein, Acinetobacter; D001565:Benzoates; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019753:Intramolecular Lyases; C021811:muconate cycloisomerase