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 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.

FIG. 1.

Catabolic reactions catalyzed by the pca and qui gene products (arrows with filled arrowheads). The biosynthetic reactions of the pathway leading to the formation of the aromatic amino acids are shown with arrows with empty arrowheads. QuiA, quinate/shikimate dehydrogenase; QuiB, dehydroquinate dehydratase; QuiC, dehydroshikimate dehydratase.

FIG. 2.

pca-qui gene region of Acinetobacter sp. strain ADP1. Black bars underneath the scheme represent hybridization probes. The results of the Northern blot experiments are summarized in the lower part of the figure. Arrows represent the largest transcripts detected with the probes indicated. Dotted lines show the possible locations of transcripts if they are ambiguous. The locations of the transcripts in this figure were determined (i) by the limits of the gene cluster (pcaI through quiA) and (ii) by the need to overlap the hybridization probes. Transcripts detected in both strain ADP1 and strain ADPU1 (ΔpcaIp) are shown with thick, dotted lines, and the respective sizes are circled.

FIG. 3.

Construction of Acinetobacter sp. strain ADPU1 with a deletion of promoter pcaIp. The small horizontal arrows indicate the three sequence repetitions of the PcaU binding site.

FIG. 4.

Reporter gene constructs to screen for promoter activity in the pcaG-quiB region and the quiX-quiA region.

FIG. 5.

Primer extension analysis upstream of quiA. Results are shown for an experiment performed with primer quiA-14844 and 10 μg of total RNA from Acinetobacter sp. strain ADP1 grown on quinate (lanes 1 and 2) or succinate (lane 3); the sequence ladder was created by using the same primer. The position of the 5′ end of the transcript is labeled +1 in the sequence, which was converted to the complementary strand (A). (B) Area upstream of quiA. Two palindromic sequence repetitions upstream of the determined 5′ end of the quiA transcript are indicated by horizontal arrows. A potential promoter sequence is indicated and compared with the consensus E. coli promoter sequence.

FIG. 6.

Transcripts from Acinetobacter sp. strain ADP1 detected with a quiX DNA probe by Northern blot analysis of total RNA (10 μg per lane). (A) Growth curve of culture in mineral medium with 20 mM pyruvate. The arrow indicates the addition of the inducer (2 mM protocatechuate). (B) Results of hybridization. Lane numbers correspond to sample numbers, and transcript sizes are given in kilobases. Arrows with empty arrowheads indicate the positions of the 16S and 23S rRNA.

FIG. 7.

Northern blot analysis of the pca-qui gene area. Samples were withdrawn during exponential growth from cultures grown in mineral medium with pyruvate 15 min after the addition of the inducer (2 mM protocatechuate) and were hybridized with the indicated probes. Results are shown for analyses of Acinetobacter sp. strain ADP1 (wild type) (A) and strain ADPU1 (ΔpcaIp) (B) All samples for each strain were taken from the same culture. The appearance of the signal was adjusted (for brightness and contrast) for the best recognition of the bands and does not represent quantitative relationships. Transcript sizes are given in kilobases. Arrows with empty arrowheads indicate the positions of the 16S and 23S rRNA.