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J Bacteriol, March 1998, p. 1512-1524, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
PcaU, a Transcriptional Activator of Genes for
Protocatechuate Utilization in Acinetobacter
Ulrike
Gerischer,1,2
Ana
Segura,1,
and
L.
Nicholas
Ornston1,*
Department of Molecular, Cellular and
Developmental Biology, Yale University, New Haven, Connecticut
06520-8103,1 and
Angewandte
Mikrobiologie, Universität Ulm, 89069 Ulm,
Germany2
Received 25 August 1997/Accepted 26 December 1997
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.
*
Corresponding author. Mailing address: Department of
Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103. Phone: (203) 432-3498. Fax: (203)
432-6161. E-mail: nick.ornston{at}quickmail.yale.edu.

Publication 17 from the Biological Transformation Center in the
Yale Biospherics Institute.

Present address: Department of Biochemistry, Consejo Superior de
Investigaciones Científicas, Estación Experimental de
Zaidín,
18008 Granada, Spain.
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