Hydroxycinnamate (hca) Catabolic Genes from Acinetobacter sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters

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American Society for Microbiology

RESUMO

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Kmr genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.

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