PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter.
The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the pca-qui-pob cluster was pobR, which encodes a transcriptional activator 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 spontaneous mutation that mapped upstream from pcaI and prevented expression of the pca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca gene expression was located in the promoter for the pca operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for Pseudomonas putida PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream from Acinetobacter pcaU contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in the pcaU-pcaI intergenic region.
Gerischer
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Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Segura
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Ornston
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D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000150:Acinetobacter / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D019343:Citric Acid / Q000378:metabolism; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D062385:Hydroxybenzoates / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D009876:Operon; D010226:Parabens / Q000378:metabolism; D010802:Phylogeny; D010957:Plasmids; D011801:Quinic Acid / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D062385:Hydroxybenzoates; D026901:Membrane Transport Proteins; D010226:Parabens; C090106:PcaK protein, Pseudomonas; C089369:PcaR protein, Pseudomonas putida; C111304:PcaU protein, Acinetobacter; C082108:PobR protein, Acinetobacter calcoaceticus; D015534:Trans-Activators; D014157:Transcription Factors; D011801:Quinic Acid; D019343:Citric Acid; C009091:protocatechuic acid; D000102:Acetyl-CoA C-Acyltransferase; C443987:3-oxoadipyl-coenzyme A thiolase; D002265:Carboxylic Ester Hydrolases; C021927:3-oxoadipate enol-lactonase; C038193:4-hydroxybenzoic acid