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

Abstract

Quinate and protocatechuate are both abundant plant products and can serve, along with a large number of other aromatic or hydroaromatic compounds, as growth substrates for Acinetobacter sp. strain ADP1. The respective genes are part of the chromosomal dca-pca-qui-pob-hca cluster encoding these pathways. The adjacent pca and qui gene clusters, which encode enzymes for protocatechuate breakdown via the ␤-ketoadipate pathway and for the conversion of quinate or shikimate to protocatechuate, respectively, have the same direction of transcription and are both expressed inducibly in response to protocatechuate. The pca genes are governed by the transcriptional activator-repressor PcaU. The mechanism governing qui gene expression was previously unknown. Here we report data suggesting the existence of a large 14-kb primary transcript covering the pca and qui genes. The area between the pca and qui genes contains no promoter activity, whereas a weak, constitutive promoter was identified upstream of quiA (quiAp). The 5 end of the quiA transcript was mapped. Northern blot analysis allowed the identification of a 12-kb transcript spanning pcaI to quiX. An analysis of the pca and qui gene transcripts in a strain missing the structural gene promoter pcaIp led to the identification of two pcaIp-independent transcripts (4 and 2.4 kb). The 2.4-kb transcript makes up about 25% of the total transcript abundance of quiA, and thus the majority of transcription of the last gene of the area is also driven by pcaIp. This report strongly supports the organization of the pca and qui genes as a pca-qui operon and, furthermore, suggests that PcaU is the regulator governing its expression.The utilization of aromatic carbon sources by Acinetobacter occurs through the ␤-ketoadipate pathway (20). This branched pathway uses two central starting compounds (protocatechuate and catechol) to funnel a large number of different aromatic or hydroaromatic monomers into the catabolic reaction sequence. One such compound is quinate, an abundant plant product (21). A set of three reactions leads to the formation of protocatechuate from either quinate or shikimate ( Fig. 1) (36, 38). The genes for these three enzymes (qui genes) (Fig. 2) have been characterized along with a gene that probably encodes a porin (10, 11). 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). The regulation of protocatechuate degradation as well as quinate utilization was studied thoroughly decade...

Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

“…Based on the sequence similarity, PraR belongs to the IclR family of transcriptional regulators, which includes PcaU and PcaR, activators of ␤-ketoadipate pathway genes in Acinetobacter baylyi ADP1 (10,20) and Pseudomonas putida PRS2000 (48), respectively, PobR, an activator of the 4HB 3-hydroxylase gene, pobA from A. baylyi ADP1 (11), as well as HmgR, a repressor of the homogentisate catabolic pathway genes of P. putida U (2). It has been reported that PCA, ␤-ketoadipate, 4HB, and homogentisate act as inducers for the PcaU, PcaR, PobR, and HmgR regulatory systems, respectively.…”

Relevance of nucleotides of the PcaU binding site from Acinetobacter baylyi

“…1b) (Dal et al, 2005). This strain is not able to grow on substrates that are degraded via protocatechuate because of the lack of the main promoter pcaIp, which drives the expression of the pca-qui operon.…”

“…This operon includes the cluster of four downstream qui genes involved in the conversion of the hydroaromatic compounds quinate and shikimate (pca-qui operon, Fig. 1; Dal et al, 2005). The pcaU gene is located upstream of this large (14 kb) operon and transcribed divergently from it.…”

Role of Acinetobacter baylyi Crc in Catabolite Repression of Enzymes for Aromatic Compound Catabolism

“…In Acinetobacter, this pathway contains two parallel branches separated in terms of enzymes, as well as regulation of expression, converting the two starting compounds protocatechuate and catechol into succinyl coenzyme A and acetyl coenzyme A (21). Protocatechuate breakdown requires six catalytic steps; the respective genes (pca genes), together with genes for one of several funneling pathways (qui genes), form a large operon (the pca-qui operon, about 14 kbp) (10). Regulation of the expression of this operon is directed from the intergenic region located upstream of this large gene cluster.…”