Multiple-level regulation of genes for protocatechuate degradation in Acinetobacter baylyi includes cross-regulation.

The bacterium Acinetobacter baylyi uses the branched beta-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the pca-qui operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter pcaIp, namely protocatechuate-dependent activation of pca-qui operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (Escherichia coli) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the pca-qui operon. The increase of the pcaU gene copy number leads to a decrease of the basal expression at pcaIp, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of pcaIp when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (cross-regulation). The same repression pattern was observed for promoter pcaUp. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (pobAp upstream of pobA, pcaIp, and pcaUp) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on pobA expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of pobA expression.

Transcriptional organization of genes for protocatechuate and quinate degradation from Acinetobacter sp. strain ADP1.

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 beta-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.

Multiple operons connected with catabolism of aromatic compounds in Acinetobacter sp. strain ADP1 are under carbon catabolite repression.

Repression of enzymes contributing to degradation of aromatic compounds via the beta-ketoadipate pathway in the presence of additional carbon sources (carbon catabolite repression) in the bacterium Acinetobacter sp. strain ADP1 is described. The phenomenon was investigated on the level of specific activity of protocatechuate 3,4-dioxygenase and p-hydroxybenzoate hydroxylase participating in catabolism of protocatechuate and p-hydroxybenzoate. Strong repression (90%) was found in cells grown on succinate and acetate in addition to the aromatic carbon source; partial derepression occurred towards the end of the logarithmic growth phase. Glucose, pyruvate, or lactate as secondary carbon sources had no repressing effect. The consumption of the aromatic substrate from the medium was delayed in the presence of acetate and succinate. The differences in specific enzyme activities were reflected at the transcript level for three operons connected to catabolism of aromatic compounds (pob, pca, van) as shown by Northern blot hybridization. Transcriptional fusions between the promoters of the pob and the pca operon identified the transcriptional level as the regulatory one. A mechanism of global regulation is postulated, which enables the organism to consume the offered carbon sources hierarchically in the most efficient manner.