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J Bacteriol, March 1998, p. 1512-1524, Vol. 180, No. 6
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.
Protocatechuate supports the growth
of diverse microorganisms (14-16, 19, 26, 27, 36, 39, 40, 42, 43,
48, 49, 57, 58, 60, 61, 64). The growth substrate may be utilized
directly or may be formed as a metabolite in the dissimilation of
compounds produced by plants in the natural environment (Fig. 1A). Representatives of the bacterial
genus Acinetobacter are ubiquitous in terrestrial habitats
(3), and metabolic pathways for utilization of the compounds
depicted in Fig. 1A have been characterized. Furthermore, structural
genes associated with the pathways have been shown to comprise the
pca-qui-pob cluster (17, 34) (Fig. 1B) in
Acinetobacter strain ADP1, an organism exceptionally amenable to genetic analysis by natural transformation (2, 10, 25,
31, 32).
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
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ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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FIG. 1.
Organization of genes associated with protocatechuate
catabolism in Acinetobacter. (A) Various growth substrates
are catabolized through pathways converging upon protocatechuate (open
box). In Acinetobacter, protocatechuate elicits expression
of all of the depicted genes except pobA, which is expressed
in response to 4-hydroxybenzoate. Protocatechuate is converted to
succinate and acetyl CoA by the consecutive actions of enzymes encoded
by the pca genes. Null mutations in pcaB cause
accumulation of the toxic metabolite carboxymuconate from
protocatechuate; exposure of cells containing the 
pcaBDK1
deletion to growth media supplemented with protocatechuate selects
colonies that fail to express pcaH and -G. Most
of these colonies are double mutants containing pcaBDK1
and a mutation in pcaH or -G (21). In
this paper, we describe another mutation, pcaP1, that blocks
the promoter for the pca operon. Characterization of DNA
flanking this mutation revealed pcaU. (B) Genes required for
catabolism of the compounds shown in panel A are grouped within the 20 kb of contiguous DNA forming the pca-qui-pob cluster in the
Acinetobacter chromosome. Directions of transcription are
indicated by dashed arrows, and the position of pcaBDK1
is shown by the grey rectangle. As described in this communication,
expression of the pca genes in response to protocatechuate
is governed by the divergently transcribed activator encoded by
pcaU (dark border). The pobA gene (10)
encodes 4-hydroxybenzoate hydroxylase and is regulated independently of
the pca and qui genes by the divergently
transcribed pobR (dark border) (9, 11).
Prior to this investigation, the only studied regulatory gene in the
Acinetobacter pca-qui-pob cluster was pobR
(9, 11, 20), which encodes a transcriptional activator
promoting expression of pobA in response to
4-hydroxybenzoate (Fig. 1). Protocatechuate was known to elicit
expression of the pca and qui structural genes (5, 6, 18) (Fig. 1), but a regulatory gene governing their transcription had not been identified. Analysis of pca
structural-gene transcription in Pseudomonas putida
(28, 46, 52) and Agrobacterium tumifaciens
(47) revealed pcaR genes encoding transcriptional activators that respond to 
-ketoadipate, an intermediate formed in
protocatechuate catabolism. -Ketoadipate does not induce
pca-encoded enzymes in Acinetobacter and has even
been used to select strains in which the enzymes are expressed
constitutively (6, 50).
Among spontaneous Acinetobacter mutant strains defective in expression of pcaH and -G were a few containing mutations outside of the pca operon (21). We describe here the characterization of one such mutation as a nucleotide substitution in the promoter of the pca structural genes. Characterization of flanking DNA revealed pcaU, a divergently transcribed gene that activates transcription of the pca genes but that is not required for their expression at levels sufficient to support slow growth.
The Acinetobacter pcaU nucleotide sequence reveals that its
product is a member of the PobR family, a subset of a sparsely represented group of transcriptional regulators in the GylR superfamily (55). Members of the PobR protein family regulate catabolic pathways for natural products from plants and include
Acinetobacter PobR (9, 11), PcaR from P. putida (28, 46, 52), and A. tumifaciens
(47). Since PcaU responds to protocatechuate and not to
p-hydroxybenzoate or -ketoadipate, it is apparent that
members of the PobR family have been adapted to respond specifically to
three different metabolite inducers in the 4-hydroxybenzoate catabolic
pathway. Remarkably conserved during this divergence were the operators
where the regulatory proteins appear to exert their different controls.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Organisms, plasmids, and growth conditions. Strains and plasmids used in this study are presented in Table 1. All organisms were grown at 37°C. Minimal medium (21) for Acinetobacter was supplemented with 10 mM succinate, 10 mM glucose, 5 mM p-hydroxybenzoate, or 5 mM protocatechuate. Escherichia coli strains were grown in Luria-Bertani medium (53). Growth media were supplemented with ampicillin (75 µg/ml), kanamycin (50 µg/ml), streptomycin (10 µg/ml), or spectinomycin (40 µg/ml) as required.
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Gene transfer by natural transformation or by conjugation. Recipient Acinetobacter cultures were grown in shake cultures overnight with 10 mM succinate. Additional succinate was added to a final concentration of 5 mM, and after 30 min of incubation, 0.1 ml of cell suspension was spread on plates containing selective medium. Solutions containing linear DNA fragments (21) or replicas of colonies (2) were placed upon cell lawns. Transformant colonies appeared after incubation for 1 or 2 days. Large plasmids were introduced into Acinetobacter by conjugation from E. coli S17-1 followed by selection for resistance to the appropriate antibiotics (54).
DNA and RNA manipulations. General DNA manipulations were performed by using published procedures (53). The alkaline lysis method (4) was used for preparation of plasmid DNA. Chromosomal Acinetobacter DNA was isolated as described previously (21). Cloned double-stranded DNA was sequenced with the Sequenase kit version 2.0 (United States Biochemical, Cleveland, Ohio) and either the supplied sequencing primers or custom-designed primers (23). Chromosomal DNA was sequenced directly as described elsewhere (21). Homology searches were conducted with the BLAST network service, and screening of nucleotide sequences for repetitions was conducted with the DNAStar software package (DNAStar Corporation, Madison, Wis.). Oligonucleotides were synthesized at Yale Medical School, New Haven, Conn.
Southern (22) and Northern (23) blot analyses were performed as described previously. DNA probes were labelled with [
-32P]dATP by using a random prime labelling kit
(Boehringer, Mannheim, Germany). Previously described procedures
(23) were used for RNA isolation and analysis of
transcription start points. The DNA probe for Northern blots of
transcripts of the pca operon was a 1,370-bp
SalI-HindIII restriction fragment containing
pcaH and -G; the DNA probe for pcaU
transcripts was a 705-bp Sau3A-EcoRI restriction
fragment from pZR9 (Fig. 2).
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Enzyme and protein determinations.
Previously described
procedures were used for assay of protocatechuate 3,4-dioxygenase
(13) and -galactosidase (41). Proteins were
separated by denaturing sodium dodecyl sulfate-gel electrophoresis as
described by Laemmli (37) with a molecular mass standard for
the low range (14.4 to 97.4 kDa) from Bio-Rad. Protein concentrations
were determined by using established procedures (38).
Gel mobility shift assays. Published procedures (1) were used to demonstrate the binding of PcaU to DNA fragments. For measurement of PcaU binding to DNA upstream of pcaU, the probe was prepared as a restriction fragment and labelled at the 3' ends with the Klenow fragment of DNA polymerase; the labelled fragments were purified by ion-exchange chromatography. A labelled fragment containing 10,000 cpm was incubated for 15 min at 30°C in 12% (vol/vol) glycerol, 300 µg of bovine serum albumin per ml, 1 µg poly(dI-dC), crude extract, and competitor DNA in a total volume of 15 µl. Samples were separated in a 4% nondenaturing acrylamide gel in low-ionic-strength buffer (6.7 mM Tris-HCl, pH 7.9; 3.3 mM sodium acetate; 1 mM EDTA). After electrophoresis, the gel was dried and exposed to X-ray film.
For measurement of PcaU binding to DNA downstream of pcaU, the DNA probe was prepared by PCR, gel purified, labelled with digoxigenin-11-dUTP with the Klenow fragment, and repurified in an acrylamide gel. Conditions for the binding reaction were the same as those described above. For the shift assay (1), 10 ng of labelled DNA was employed in each incubation. After electrophoresis for 1 h in a 4% nondenaturing acrylamide gel in low-ionic-strength buffer, DNA was transferred by capillarity with 10× saline sodium citrate buffer to a nylon membrane over 5 h and fixed for 2 min with UV light. The membrane was soaked overnight in washing buffer, and chemiluminescent detection was achieved by following the instructions of the manufacturer (Genius System; Boehringer Mannheim).Introduction of designed mutations into bacterial strains.
Acinetobacter strain ADP92 was formed by introduction of the
SmrSpcr cassette from plasmid pHP45 into
the sole XhoI site of pcaU in plasmid pZR9. The
SmrSpcr cassette was removed from pHP45
by cleavage with EcoRI and PstI. The ends of both
DNA preparations were filled in with the Klenow fragment, ligated, and
transformed into E. coli DH5. Clones with a plasmid
containing the cassette within pcaU were selected on Luria-Bertani medium supplemented with streptomycin and spectinomycin. The resulting plasmid, designated pZR91 (Fig. 2), was cut with EcoRI, releasing a 2.7-kb DNA fragment. The DNA fragment was
eluted from an agarose block after preparative agarose gel
electrophoresis and used as a donor in transformation of the wild-type
Acinetobacter strain ADP1. Selection for streptomycin and
spectinomycin resistance produced strain ADP92.
. The presence of the desired deletion in pZR33 was confirmed by
restriction analysis and DNA sequencing. After digestion of pZR33 with
HindIII and SstI, DNA containing the deletion
was introduced into wild-type Acinetobacter strain ADP1 by
natural transformation. Recombinant colonies on succinate plates were
identified by colony hybridization with the
NcoI-MluI fragment as a probe to screen for
clones in which it was lacking. The presence of the
NcoI-MluI deletion in strain ADP331 was confirmed by PCR.
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Nucleotide sequence accession number. The nucleotide sequence data presented in this paper has been deposited with GenBank under accession no. U04359.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The pcaP1 mutation is upstream of pcaI and
blocks expression of pca structural genes.
Strains
blocked in expression of pcaH and -G, structural
genes for protocatechuate 3,4-dioxygenase, can be selected by demanding growth of organisms carrying the pcaBDK1 mutation
(Fig. 1) with succinate in the presence of protocatechuate. A previous
study (21) showed that most of the selected
strains contain, in addition to pcaBDK1, mutations in
pcaH and -G. As described in the legend to Fig.
2, strain ADP6126 contains an exceptional mutation, pcaP1, which lies outside the chromosomal region occupied by the
pca structural genes.
Sequencing of DNA replacing pcaP1 revealed both pcaU and a 282-bp pcaU-pcaI intergenic region. The nucleotide sequence of the Sau3A insert in pZR9 overlapped the known sequence of the pZR1 EcoRI insert by 4,698 bp and contained 705 bp of previously uncharacterized DNA. Sequencing the latter DNA segment revealed a portion of a divergent open reading frame with two possible translational origins, either 282 or 294 bp from the start of pcaI (Fig. 2). In order to complete the nucleotide sequence of the newly discovered open reading frame, the 705-bp Sau3A-EcoRI fragment was used as a probe in Southern hybridization of Acinetobacter DNA restriction fragments to find an endonuclease that generated a fragment of appropriate size and location for further analysis. On the basis of this information, a 7-kb HpaII fragment was cloned into the AccI site of pUC19. The insert of the new plasmid, pZR15, extends through and beyond the newly characterized chromosomal region. Sequencing of pZR15 subclones (Fig. 3) revealed an open reading frame, designated pcaU, encoding a protein of either 274 or 278 residues (Fig. 4) that is a member of the PobR family of transcriptional activators (29). Among known members of this family, Acinetobacter PobR (9, 11), the transcriptional activator of pobA, is most similar to PcaU, to which it displays 54% amino acid sequence identity (Fig. 4). Among other known representatives of the family, the closest evolutionary affinity was exhibited by P. putida PcaR (52), which displays 31% amino acid sequence identity to Acinetobacter PcaU.
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Two open reading frames apparently associated with -oxidation of
acyl CoA thioesters are located downstream from pcaU.
The
newly obtained nucleotide sequence revealed a portion of an open
reading frame potentially encoding a polypeptide of 340 residues (Fig.
5). This sequence corresponds to the
carboxyl-terminal portion of an enzyme family of flavin adenine
dinucleotide-containing acyl coenzyme A (CoA) dehydrogenases generally
associated with the oxidation of the fatty acid thioesters. A
neighboring open reading frame (Fig. 5) potentially encodes an
81-residue protein that is a member of a family of enoyl-CoA
dehydratases also associated with metabolic pathways for fatty acyl CoA
oxidation. Thus, both open reading frames appear to be associated with
a physiological function demanding the -oxidation of acyl CoA
thioesters, metabolic steps that are not associated with
protocatechuate catabolism.
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Expression of PcaU from its cloned gene in E. coli.
The
open reading frame inferred to be pcaU encodes a polypeptide
with a predicted molecular mass of either 31,610 or 31,092 Da. A cloned
DNA fragment containing the gene was used to demonstrate synthesis of
the corresponding protein in E. coli. The
HindIII-HpaII restriction fragment containing
pcaU was placed under the control of the lac
promoter in the broad-host-range plasmid pWH661+, giving
rise to pZR22; pZR18 contains the same insert in the opposite
orientation (Fig. 3). As shown in Fig. 6,
E. coli DH5(pZR22) expresses substantial quantities of a
29,000-Da protein at a level that is below the level of detection in
E. coli DH5(pZR18). Insertion of lacZ into the
XhoI site of pcaU disrupts the gene in pZR26, which in other respects is identical to pZR22 (Fig. 3). Figure 6
demonstrates that the E. coli strain containing the
pcaU::lacZ insert replaces the protein
corresponding to PcaU with a very large protein likely to correspond to
LacZ.
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PcaU binds to DNA containing a consensus operator sequence located
between the pcaU and pcaI promoters.
Extracts of E. coli DH5(pZR22) containing PcaU were used
to observe binding of the protein to DNA fragments containing a portion
of the pcaU-pcaI intergenic region. The initial probe for
gel shift assays was the 417-bp XmnI-HpaII
fragment prepared from pZR18 (Fig. 3). This DNA was divided into a
201-bp HpaII-EcoRI fragment containing a portion
of pcaI and a 216-bp XmnI-EcoRI fragment containing a portion of pcaU (Fig. 3). The latter
fragment and the 417-bp fragment bound PcaU; the 201-bp
HpaII-EcoRI fragment did not. Results obtained
with the XmnI-EcoRI fragment are shown in Fig.
7. Two shifted bands were observed (Fig.
7). The addition of protocatechuate to the binding reaction did not
significantly change the observed protein-DNA interaction.
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Identification of consensus operator sequences.
The 216-bp
XmnI-EcoRI fragment that is bound by PcaU
contains a 19-bp segment that displays 84% nucleotide sequence
identity to the Acinetobacter pobR operator. Comparison of
these sequences with operators governing expression of P. putida
pca genes in response to PcaR revealed a consensus sequence which
exhibits identity in at least 16 of the 19 residues in each comparison (Fig. 8). On the basis of this evidence,
it is reasonable to propose that the newly sequenced 19-bp segment
represents a portion of the pcaU operator. Thus, it is
apparent that three chemically different inducers
(p-hydroxybenzoate, protocatechuate, and -ketoadipate, respectively) exercise precise transcriptional control through the
interaction of homologous proteins (PobR, PcaU, and PcaR, respectively) with DNA segments containing closely similar nucleotide sequences (Fig. 8).
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Demonstration of a PcaU-binding site directly downstream from pcaU. The availability of a consensus nucleotide sequence for DNA that may bind transcriptional regulators in the PobR family prompted a scan of the 20-kb Acinetobacter pca-qui-pob cluster for additional sequences displaying identity to the consensus sequence at more than 15 of 19 aligned residues. The survey revealed only one additional sequence, a DNA segment directly downstream from pcaU that differed from the consensus sequence in only 2 of 19 positions (Fig. 8). More-extensive comparison of the downstream sequence with the putative operator upstream from pcaU revealed that 33 of 43 aligned residues were identical after the introduction of a gap corresponding to one nucleotide in the downstream sequence (Fig. 5). This observation warranted a direct determination of the ability of PcaU to bind to DNA downstream from pcaU, and such evidence (Fig. 9) was obtained with a 300-bp PCR fragment containing the downstream sequence (Fig. 4). Thus, it appears that there are two binding sites for PcaU, one upstream and one downstream from pcaU.
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PcaU is an activator of pca structural
genes.
The physiological function of pcaU was explored
by the preparation of strains carrying mutated forms of the gene. One
of these, strain ADP92, contains the mutation
pcaU1::SmrSpcr (Fig.
2). This insertion would be expected to terminate both transcription
and translation of pcaU at the XhoI site within its presumed helix-turn-helix DNA binding motif (Fig. 4). The other
mutant strain, ADP331, was prepared from plasmid pZR33 (Fig. 3) and
lacks the NcoI-MluI restriction fragment (Fig. 4)
corresponding to two-thirds of pcaU. Both ADP92 and ADP331
expressed the full set of pca structural genes, as judged by
their ability to grow, albeit more slowly than wild-type cells, on
plates containing p-hydroxybenzoate as the carbon source.
SmrSpcr and
recA100::Tn5.
Repression of pcaU transcription is relieved by
protocatechuate.
In order to examine the influence of
pcaU on its own expression, a promoterless
lacZ:Kmr cassette was introduced into the
XhoI site of pZR92, giving rise to the transcriptional
fusion pcaU::lacZ in pZR28 (Fig. 2).
Expression of the lacZ insert from the pcaU
promoter was examined in cells containing
recA100::Tn5. There was no functional
pcaU in strains containing the chromosomal
pcaU1::SmrSpcr
mutation, and during growth with succinate these organisms expressed lacZ from the plasmid at levels corresponding to 1,100 Miller units; the addition of protocatechuate to the growth medium
increased these levels slightly to 1,400 Miller units. In the presence
of wild-type chromosomal pcaU, expression of lacZ
from the plasmid corresponded to levels between 300 and 350 Miller
units during growth with succinate. When protocatechuate was added to
the growth medium of these cells, lacZ expression increased
to levels corresponding to 1,200 Miller units. Thus PcaU
appears to cause a three- to fourfold repression of pcaU
expression under these conditions, and this repression is relieved by
protocatechuate.
Regulation by protocatechuate is exerted at the transcriptional level; the locations of the transcriptional starts of pcaI and pcaU. Primer extension (Fig. 10A) revealed the beginning of the pcaI transcript, 119 bp upstream from the gene and 6 bp distal to the intergenic EcoRI site (Fig. 11). Primer extension (Fig. 10B) also showed that the pcaU transcript starts 31 bp upstream from pcaU (Fig. 11) and 132 bp from the transcriptional start for pcaI (Fig. 11). The starts of both transcripts are apparent in extracts of cells grown under inducing conditions and below detectable levels in uninduced cells (Fig. 10). This finding allows the conclusion that protocatechuate stimulates transcription of both pcaU and the pca operon. Further evidence that protocatechuate increases transcription of pcaU in wild-type cells emerged from Northern blots showing the formation of transcripts corresponding to both pcaU and the pca operon in response to growth with either p-hydroxybenzoate or protocatechuate (results not shown).
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The location of pcaP1 is consistent with its being a
promoter mutation.
The nucleotide sequence of DNA containing
pcaP1 showed a single nucleotide substitution consistent
with a change at the 
35-bp promoter region for the pcaI
transcript (Fig. 11).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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An overview of the genetic region containing pcaU. The available evidence supports the conclusion that PcaU activates transcription of the pca operon by binding to an operator between pcaI and pcaU (Fig. 11). The first indication of where transcriptional regulation is exerted upstream of pcaI was given by the phenotype conferred by pcaP1, which mapped in the intergenic region. The mutation blocks expression of pca structural genes, and its location, 35 bp upstream from the position where pcaI transcription begins, suggests that it blocks promoter function (Fig. 11).
Translation of the open reading frame designated pcaU begins either 282 or 294 bp upstream from the translational start of pcaI, and the pcaI-pcaU intergenic region exhibits considerable complexity. Between the transcriptional starts of pcaI and pcaU is a presumptive pca operator which bears striking nucleotide sequence similarity to the operator governing pob expression (Fig. 11). A scan of the 20-kb pca-qui-pob region revealed that directly downstream from pcaU is an additional nucleotide sequence resembling the pcaU operator (Fig. 5). This raised the possibility that PcaU may bind to two different locations, one in the pcaI-pcaU intergenic region and the other downstream from pcaU. This inference was supported by direct measurement of PcaU binding to the two genetic regions (Fig. 8 and 9). Simultaneous binding to both regions by PcaU could allow the protein to regulate its own synthesis by looping (56) of DNA containing pcaU. An intergenic region of 404 bp separates pcaU from open reading frames, here designated orf1 and orf2, that, on the basis of nucleotide sequence comparison, appear to be associated with the-oxidation of CoA thioesters of carboxylic acids (Fig. 5). Whatever
the natural substrates of these enzymes are, they are not
metabolites formed in the catabolism of protocatechuate, quinate, or
p-hydroxybenzoate. Thus, the chromosomal cluster of
catabolic genes including pca-qui-pob extends to additional
genes with as yet unknown function.
Transcriptional controls exerted by PcaU. PcaU is a transcriptional activator of the pca operon. Inactivation of pcaU lowers induced expression of pcaH and -G by 80 to 90% and severely reduces the rate of growth of cells with p-hydroxybenzoate. When expressed from a plasmid in trans, pcaU can foster a 10-fold induction of pca structural genes in response to protocatechuate. Analysis of pcaU transcriptional fusions demonstrated that the wild-type gene product represses expression of its gene about threefold, and this repression is relieved by protocatechuate. The induction of transcripts for both pcaU and the pca operon in response to protocatechuate was demonstrated by primer extension and by Northern blot analysis.
The level of expression of the pca operon in the absence of PcaU is fairly high. Two possible factors may contribute to the background expression of the pca operon. First, there may be additional transcriptional activators that to some extent mimic the effect of PcaU in Acinetobacter. Second, the structure of the pca operon promoter may allow a significant level of constitutive transcription. In any case, the observed background of gene expression fits with the notion of regulatory noise, the hypothesis that transcriptional controls allowing a low level of background expression are candidates well-suited to being borrowed in the evolution of new regulatory mechanisms (8).Comparison of PcaU and PobR. PcaU contains either 274 or 278 amino acid residues, and most of these are in a sequence closely resembling the amino acid sequence of PobR. Introduction of a single gap into the PcaU sequence allows an alignment in which PcaU and PobR exhibit 54% identity over 255 amino acid residues (Fig. 4). The similarity extends through the presumed helix-turn-helix DNA-binding region (Fig. 4) where 13 of 22 amino acid residues are identical. The proteins bear no significant sequence similarity in their NH2-terminal regions which are highly charged.
Comparison of the pcaI-pcaU and pobA-pobR intergenic regions. The common ancestry of PcaU and PobR can be inferred from the similarity of their amino acid sequences (Fig. 4), and this conclusion is strengthened by the resemblance of the operators to which the respective proteins probably bind (Fig. 11). Perhaps more remarkable are the variations achieved by the two regulatory systems during their evolutionary divergence. At the phenotypic level, control exercised by PcaU appears to be lax, whereas regulation exerted by PobR is quite tight: in the absence of either p-hydroxybenzoate or a functional pobR gene, pobA is not expressed at detectable levels. Knockout mutations in pobR completely block growth with p-hydroxybenzoate, whereas knockout mutations in pcaU do not prevent slow growth supported by the pca structural genes. Differences extend to control of regulatory gene expression: pcaU and pobR are repressed by their respective protein products, but repression exerted by PcaU is relieved by its effector, protocatechuate, whereas p-hydroxybenzoate, the PobR effector, does not relieve the repression exerted by this protein upon its gene (11).
The pobA-pobR intergenic region is much more tightly structured than the pcaU-pcaI intergenic region. Only 21 bp separate the transcriptional and translational starts of pobA (Fig. 11). In contrast, the transcriptional and translational starts of pcaI are separated by 118 bp of DNA with unknown function (Fig. 11). The pca operator lies between the promoters for pcaI and pcaU (Fig. 11). In contrast, extremely tight organization superimposes the pobA and pobR promoters upon each other and places the pob operator between the transcriptional and translational starts of pobR (Fig. 11).DNA arrangements and rearrangements during evolution.
The
overall pattern that emerges from a comparison of pcaU and
pobR is that of conservation of similar transcriptional
activators and consensus operators against a background of extensive
evolutionary divergence in the Acinetobacter pca-qui-pob
gene cluster. The common themes are maintained in a broader biological
context. PcaU and PobR are members of a somewhat sparsely represented
transcriptional-activator family that has been called upon during
evolution of the -ketoadipate pathway (9, 29, 47). The
closest known relatives of Acinetobacter PcaU and PobR are P. putida PcaR (52) and
Erwinia chrysanthemi KdgR (45). All four proteins
respond to chemically different inducing metabolites (Fig.
12). Three of the proteins (PcaU, PobR, and PcaR) are associated with the same physiological function, growth
with p-hydroxybenzoate through the -ketoadipate pathway. Thus, it appears that a critical stage in the evolution of controls for
the -ketoadipate pathway was the selection of an ancestor of the
PobR family as a transcriptional activator. Subsequent events resulted
in modified transcriptional regulators that respond to different
inducers.
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-ketoadipate pathway interact with closely similar
operators, and the consensus nucleotide sequences in these operators
are not apparent in the operators with which KdgR interacts (44,
45) (Fig. 12). The operators associated with the -ketoadipate pathway are closely similar to the E. coli glpR operator
(63) which interacts with a transcriptional activator
(7) from the separate DeoR (59) family of
transcriptional regulators (Fig. 12). The close similarities of the
operator sequences might be the product of convergent evolution.
Alternatively, it is possible that DNA rearrangements may have produced
novel combinations of operators and regulatory proteins during the
evolution of transcriptional controls.
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ACKNOWLEDGMENTS |
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This research was supported by grants from the Army Research Office and the National Science Foundation. U.G. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. A.S. was supported by a postdoctoral fellowship from the Spanish Ministerio de Educacion y Ciencia.
We are indebted to San Tran and Stephanie Haus for technical assistance, to David Young for assistance in the identification of a consensus sequence, and to Wayne Coco and David D'Argenio for improvements made to the manuscript.
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FOOTNOTES |
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* 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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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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