Genes for Chlorogenate and Hydroxycinnamate Catabolism ( hca ) Are Linked to Functionally Related Genes in the dca-pca-qui-pob-hca Chromosomal Cluster of Acinetobacter sp. Strain ADP1

Abstract

Hydroxycinnamates are ubiquitous in the environment because of their contributions to the structure and defense mechanisms of plants. Additional plant products, many of which are formed in response to stress, support the growth of Acinetobacter sp. strain ADP1 through pathways encoded by genes in the dca-pca-qui-pob chromosomal cluster. In an appropriate genetic background, it was possible to select for an Acinetobacter strain that had lost the ability to grow with caffeate, a commonly occurring hydroxycinnamate. The newly identified mutation was shown to be a deletion in a gene designated hcaC and encoding a ligase required for conversion of commonly occurring hydroxycinnamates (caffeate, ferulate, coumarate, and 3,4-dihydroxyphenylpropionate) to thioesters. Linkage analysis showed that hcaC is linked to pobA. Downstream from hcaC and transcribed in the direction opposite the direction of pobA transcription are open reading frames designated hcaDEFG. Functions of these genes were inferred from sequence comparisons and from the properties of knockout mutants. HcaD corresponded to an acyl coenzyme A (acyl-CoA) dehydrogenase required for conversion of 3,4-dihydroxyphenylpropionyl-CoA to caffeoyl-CoA. HcaE appears to encode a member of a family of outer membrane proteins known as porins. Knockout mutations in hcaF confer no discernible phenotype. Knockout mutations in hcaG indicate that this gene encodes a membrane-associated esterase that hydrolyzes chlorogenate to quinate, which is metabolized in the periplasm, and caffeate, which is metabolized by intracellular enzymes. The chromosomal location of hcaG, between hcaC (required for growth with caffeate) and quiA (required for growth with quinate), provided the essential clue that led to the genetic test of HcaG as the esterase that produces caffeate and quinate from chlorogenate. Thus, in this study, organization within what is now established as the dca-pca-qui-pob-hca chromosomal cluster provided essential information about the function of genes in the environment.

Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1

“…Consisting of variations on a phenolic ring with a propenoate side chain, hydroxycinnamates play versatile roles as components or precursors in plant architecture and defense (9,48). The ability of bacteria to utilize diverse hydroxycinnamates as sources of carbon and energy is widely distributed among microbial groups (1,8,29,32,39,46,47). As is frequently the case with aromatic compounds (44), hydroxycinnamates tend to be toxic to bacterial cells (38).…”

“…One cluster contains genes for pathways of dicarboxylate (dca), protocatechuate (pca), quinate or shikimate (qui), and p-hydroxybenzoate (pob) catabolism (35). Characterization of DNA beyond the pob genes in this strain led to the identification of all of the hca genes involved in hydroxycinnamate utilization (37,46). The pca, qui, pob, and hca genes are functionally related in that quinate, shikimate, p-hydroxybenzoate, and hydroxycinnamates are all degraded via the protocatechuate branch of the ␤-ketoadipate pathway.…”

“…HcaB, an aldehyde dehydrogenase, transforms the aldehyde to a carboxylated derivative that serves as a substrate for downstream catabolic pathways. A fourth gene, hcaG, encodes an esterase that hydrolyzes chlorogenate to quinate and caffeate (46). An exception to the proximity of related genes is the situation for the van genes for vanillate degradation, which are unlinked to the hca region (43).…”

“…In Acinetobacter strains, dissimilation of the latter two compounds occurs via the intermediates p-hydroxybenzoate and vanillate, respectively ( Fig. 1) (8,37,46). The hca genetic cluster consists of two groups of genes: hcaABCDEFG and the divergently transcribed hcaKR (Fig.…”

Transcriptional Organization of Genes for Protocatechuate and Quinate Degradation from Acinetobacter sp. Strain ADP1

“…Together with the adjacent pca genes (Fig. 2) encoding all of the enzymes for protocatechuate conversion into succinyl-coenzyme A (CoA) and acetyl-CoA, they are part of the dcapca-qui-pob-hca chromosomal cluster of genes and have coding capacity for the catabolic conversion of several plant products (dicarboxylic acids, protocatechuate, quinate, phydroxybenzoate, and hydroxycinnamates), all of which, with the exception of the dicarboxylic acids, are channeled into the ␤-ketoadipate pathway (25,30,35). All 13 genes of the pca and qui gene cluster are transcribed in the same direction, with the exception of the regulator gene pcaU (15).…”