PCAU, A TRANSCRIPTIONAL ACTIVATOR OF GENES FOR PROTOCATECHUATE UTILIZATION IN ACINETOBACTER

The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that con vert protocatechuate to citric acid cycle intermediates, Directly down stream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocate chuate. Prior to this investigation, the only known regulatory gene In the pca-qui-pob cluster was pobR, which encodes a transcriptional act ivator that responds to p-hydroxybenzoate and activates transcription of pobA. The pca and qui genes were known to be expressed in response to protocatechuate. but a protein that mediated this induction had not been identified, This study was initiated by characterization of a sp ontaneous mutation that mapped upstream from peal and prevented expres sion of the pea genes, Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mut ation revealed a 282-bp intergenic region and a divergently transcribe d open reading frame, designated pcaU. Downstream from pcaU are two op en reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters, Inactivation o f pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant c ells with protocatechuate, PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containi ng a sequence identical in 16 of 19 nucleotide residues to a segment o f the pob operator, Further similarity of the two regulatory systems i s indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU, The pob and pea systems were shown to di ffer, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the sine of the intergenic region, and the tightness of transcriptional control, The spontaneous mutation blocking pca gene expression was located in t he promoter far the pca operon, The 19-nucleotide residue operator seq uences were shown to be parts of a consensus associated with transcrip tional activation of genes associated with protocatechuate catabolism, Two different binding sites for Pseudomonas patida PcaR differ from t he consensus in only a single nucleotide residue, and DNA directly dow nstream from Acinetobacter pcaU contains a 19-bp segment differing fro m the consensus in only two residues, PcaU was shown to bind to DNA co ntaining this segment as well as to the DNA in the pcaU-pcaI intergeni c region.