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Journal of Bacteriology, April 2000, p. 2253-2261, Vol. 182, No. 8
Department of Microbiology and Molecular
Genetics, University of Texas Medical School, Houston, Texas
77030,1 and Department of Microbiology
and Parasitology, University of Queensland, Brisbane, Queensland
4072, Australia2
Received 18 October 1999/Accepted 18 January 2000
The transcription factor PpsR from the facultative photoheterotroph
Rhodobacter sphaeroides is involved in repression of
photosystem gene expression under aerobic growth conditions. We have
isolated a number of spontaneous mutations as well as constructed
directed mutations and deletions in ppsR. Repressor
activities and the oligomeric state of the wild-type and mutant
proteins were assayed. Our results suggest that the wild-type
PpsR exists in cell extracts as a tetramer. Analysis of the PpsR
mutants confirmed that the carboxy-terminal region of PpsR (residues
400 to 464) is involved in DNA binding. The central region of the
protein (residues 150 to 400) was found to contain two PAS domains
(residues 161 to 259 and 279 to 367). PAS domains are ubiquitous
protein modules involved in sensory transduction as well as in
protein-protein interactions. All spontaneously isolated mutations,
which significantly impaired repressor activity and which mapped
outside the DNA binding region, were positioned in the PAS domains.
None of these, however, affected the overall oligomeric state.
This implies that the conformation of the PAS domains within the
tetramer is critical for repressor activity. Upstream of the first PAS
domain resides a putative glutamine-rich hinge (residues 127 to 136)
that connects the first PAS domain to the amino-terminal region
(residues 1 to 135). The role of the amino terminus of PpsR is not
obvious; however, extended deletions within this region abolish
repressor activity, thus suggesting that the amino terminus is
essential for structural integrity of the protein. We present a model
of the domain architecture of the PpsR protein according to which PpsR
is comprised of three regions: the carboxy terminus responsible for DNA
binding, the central region primarily involved in protein
oligomerization and possibly signal sensing, and the amino terminus of
unknown function. This model may prove useful for determining the
mode of PpsR action.
The transition of the facultative
photoheterotroph Rhodobacter sphaeroides
from an aerobic to an anaerobic environment requires, among other
things, a significant increase in expression of the photosystem genes.
These genes encode structural and assembly proteins of the light
harvesting and reaction center complexes as well as proteins involved
in biosynthesis of the photopigments, bacteriochlorophyll
(bch genes) and carotenoids (crt genes).
When oxygen tension decreases below certain threshold level, several major regulatory circuits are recruited to increase photosystem gene
expression and hence photosystem production (19, 30). One of
these circuits involves derepression of the bch and
crt genes, as well as the puc operon, encoding
light harvesting II complex proteins, by the inactivation of the
repressor PpsR.
The PpsR repressor blocks transcription by binding to the sequences
TGTN12ACA (where N is nucleotide), which are positioned either downstream of or overlapping with the promoters of a number of
bch and crt genes as well as the puc
operon (9, 20, 22). Under aerobic conditions, expression of
these genes is low, while under semiaerobic or anaerobic conditions,
repression is greatly decreased or abolished. The extent of
PpsR-mediated repression is controlled at the level of repressor
activity rather than PpsR protein abundance (10). Very
little is known about the mechanism(s) of such control, although we
have presented evidence that PpsR most likely does not interact with
oxygen directly. Furthermore, PpsR retains partial repressor
activity under fully anaerobic, photosynthetic conditions, and this
activity diminishes with decreased light intensity (10).
Therefore, we were led to suggest that instead of sensing primary
environmental signals, i.e., oxygen tension and light intensity, PpsR
may sense cellular redox poise or the redox state of a specific redox
carrier (12). In accord with this hypothesis, in vitro DNA
binding by CrtJ, a PpsR homolog from a related bacterium,
Rhodobacter capsulatus, was shown to depend on
the redox compounds present in the buffer (21).
Initial analysis of PpsR/CrtJ revealed no significant similarities to
other proteins in the databases, except for the helix-turn-helix (HTH)
motif at the carboxy terminus, which was proposed to be involved in DNA
binding (20). More recently, a PAS domain was identified in
these proteins (24). PAS domains are universal modules for
signal sensing and transduction in proteins from Bacteria, Archaea, and Eucarya. Many such proteins are
involved in cellular responses to light, oxygen, or changes in redox
poise (24, 28).
We undertook a mutational analysis of ppsR to gain greater
insight into the structural organization and possible mode of action of
the PpsR repressor. To isolate mutations in ppsR, we took
advantage of an earlier observation that all spontaneously appearing
suppressors of the R. sphaeroides appA null mutation map
to the ppsR gene (10). AppA is a redox regulator
of photosystem gene expression that antagonizes PpsR repressor activity
in vivo by an as yet unknown mechanism (7, 8, 10). The
spontaneous mutations were cloned, and their repressor activities were
assayed. We also constructed a number of directed mutations. In
addition to PpsR activity, we determined the oligomeric state of the
wild-type and mutant proteins. Based on analyses of mutants and protein sequence motifs within PpsR, we propose a model domain architecture of
this protein which could help in elucidating its mechanism of action.
Bacterial strains and plasmids.
Strains and plasmids used in
this work are listed in Table 1.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Domain Structure, Oligomeric State, and Mutational Analysis
of PpsR, the Rhodobacter sphaeroides Repressor of
Photosystem Gene Expression
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this work
Microbiological and genetic techniques. Escherichia coli strains were grown at 37°C on Luria-Bertani medium (17) supplemented, where required, with the following antibiotics at the indicated final concentrations: tetracycline, 10 µg/ml; ampicillin, 100 µg/ml; streptomycin and spectinomycin, 25 µg/ml each.
R. sphaeroides strains were grown anaerobically at 30°C on Sistrom's medium A (2) containing succinate as the carbon source in fully filled screw-cap tubes which were illuminated with light at 10 W/m2. Paracoccus denitrificans liquid cultures (10 ml) were grown on the same medium as R. sphaeroides in 125-ml flasks under vigorous shaking. Antibiotics for R. sphaeroides and P. denitrificans were used, where appropriate, at the indicated final concentrations: tetracycline, 1 µg/ml; streptomycin plus spectinomycin, 50 µg/ml each. Conjugation was performed essentially as described previously (3). To introduce plasmids of interest into various R. sphaeroides and P. denitrificans strains, biparental matings (with E. coli S17-1 as a donor) or triparental [with E. coli DH5
phe(pRK2013) as
a helper strain] were used (Table 1).
Spectrophotometric assays.
R. sphaeroides cell
extracts were obtained by sonication of photosynthetically grown cells
and assayed as described previously (16), using samples
containing equal amounts of protein. Photosynthetic growth of R. sphaeroides strains was monitored using a Klett-Summerson photometer with filter no. 66 and is expressed in Klett units, where 1 Klett unit equals approximately 107 cells
ml
1.
-Galactosidase assays.
In aerobically grown P. denitrificans, -galactosidase was assayed in whole cells
treated with chloroform and sodium dodecyl sulfate as described earlier
(9). -Galactosidase activity is expressed in Miller
units, where 1 Miller unit corresponds to 1 µmol of
o-nitrophenyl--galactoside hydrolyzed per min per unit of
optical density of culture at 600 nm (OD600). All assays were performed at least twice with standard deviations not exceeding 15%.
DNA manipulations and sequence analysis. Standard recombinant DNA techniques (17) and molecular biological enzymes and reagents were used according to the specifications of the manufacturers. DNA sequencing of the ppsR mutant alleles was performed on an ABI 377 automatic DNA sequencer (Applied Biosystems) at the DNA Core Facility of the Department of Microbiology and Molecular Genetics.
Isolation of the ppsR mutations. Mutations were isolated as described earlier (10). Briefly, the R. sphaeroides AppA null mutant, APP11, was plated under photosynthetic conditions, and photosynthesis-competent pseudorevertants were isolated and streak purified. ppsR mutant alleles were either cloned from minilibraries made from chromosomal DNA of the isolated clones (10) or amplified by high-fidelity PCR using Pfu polymerase and primers flanking the ppsR coding sequence. All ppsR alleles were sequenced using ppsR-specific primers at the DNA Core Facility of the Department of Microbiology and Molecular Genetics. The 1.5-kb NsiI-NcoI fragments containing mutant ppsR alleles were cloned into vector pRK415 in the identical fashion to create plasmids designated pPS# (# represents the number of a given mutation).
Purification of a GST-PpsR fusion protein.
The 1.6-kb
BspEI-XmaI fragment containing the
ppsR gene was cloned into the XmaI site of vector
pGEX-2TK downstream of the gene encoding glutathione S-transferase
(GST) to create plasmid pGpps. The GST-PpsR fusion protein was
expressed in E. coli JM109(pGpps). Optimal production of
a soluble GST-PpsR fusion occurred when mid-log cells
(OD600 = 0.6), grown at 25°C, were induced with 0.1 mM isopropyl--D-thiogalactopyranoside for 8 h. Two
liters of E. coli was grown for production of GST-PpsR.
Harvested cells were disrupted in a French cell pressure unit. The
soluble extract was then fractionated using a DEAE ion-exchange column
(80-ml bed volume) equilibrated with 50 mM Tris-HCl (pH 7.5) (elution buffer). GST-PpsR was eluted using a 0 to 500 mM NaCl elution buffer,
and peak fractions of GST-PpsR were obtained at about 270 mM NaCl.
Pooled fractions containing GST-PpsR were charged onto a
glutathione-Sepharose column (bed volume, 15 ml). After washing with 80 mM phosphate (pH 7.7)-0.4% n-octylglucoside, the GST-PpsR
fusion was eluted with 5 mM glutathione in 80 mM phosphate (pH
7.7)-0.4% n-octylglucoside. The protein was judged to be
pure as indicated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis; we obtained a single polypeptide of 76 kDa,
close to the expected size of a GST-PpsR fusion.
Production of PpsR-specific antibodies. The purified GST-PpsR protein was used to immunize chickens. Antibodies were obtained from egg yolks by ammonium sulfate precipitation (27). PpsR-specific antibodies were purified by immunoaffinity chromatography; 7.5 mg of cell extract of E. coli JM109(pRK415) was coupled to a 1-ml HiTrap N-hydroxysuccinimide column (Amersham Pharmacia Biotech). The crude antibody mixture was then run through the column, and protein fractions that did not bind to the column were collected and tested for anti-PpsR reactivity.
Western blotting and Ferguson plot analysis. Western blot analyses were performed by standard protocols. PpsR-specific antibodies were used in a 1:100 ratio as a primary antibody; a mouse anti-chicken secondary antibody coupled to alkaline phosphatase was used for development of the immunoblots.
The native molecular mass of PpsR was determined by Ferguson plot analysis of the relative mobility of the native protein and a series of protein standards (13). Electrophoretic analysis was performed by Western blotting at six different acrylamide concentrations between 7.5 and 10% (30:0.8 acrylamide/bisacrylamide). After the running of each gel, the proteins were transferred to nitrocellulose. The protein standards were stained with Ponceau S and washed with water. PpsR was detected in the cell extracts contained on the nitrocellulose membrane by Western blotting. The protein standards used were lactalbumin (14 kDa), bovine serum albumin (66 kDa), dimethyl sulfoxide reductase (82 kDa), amylase (180 kDa), and urease (262 kDa). The log10 Rf for each protein was plotted as a function of polyacrylamide gel concentration. The slope of each plot gave the retardation coefficient for each protein (25). This slope was then plotted as a function of native Mr, and from this calibration curve the native Mr of PpsR was determined.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation and characterization of R. sphaeroides PpsR mutants. We have previously described (10) the procedure for isolating mutations in the R. sphaeroides ppsR gene and have mapped some of these mutations. The procedure is based on selection of photosynthesis-competent pseudorevertants of an AppA null mutant, which itself is impaired in photosynthetic growth. The ppsR gene is the only site found to date for suppressor mutations, i.e., overcoming the initial AppA null phenotype. Using this procedure, we isolated ppsR mutations in addition to those previously described (Table 1). Here, we present a detailed characterization of the isolated ppsR mutations as a means to better understanding of PpsR structure and function.
We tested for all of the mutants both photosynthetic growth and the abundance of photosynthetic complexes, traits which appear to correlate with the residual activity of the mutant PpsR repressor. The mutants of one class of suppressors (Table 2, class 1), were similar to the AppA PpsR double null mutant, APS1, in their rapid transition from aerobic to anaerobic photosynthetic growth conditions (Fig. 1A, strain PS40). These mutants accumulated photosynthetic complexes to high abundance (Fig. 1B, strain PS40) and, like mutant APS1, were genetically unstable when grown under aerobic conditions, apparently because of accumulation of photosynthetic complexes in the presence of oxygen (data not shown). The mutants of class 3 (Table 2) showed only slight improvement in the transition from aerobic to anaerobic photosynthetic growth compared to the parent AppA null mutant, APP11 (Fig. 1A, strain PS23). These contained only low levels of photosynthetic complexes (Fig. 1B, strain PS23). The phenotypes of the remaining mutants, placed in class 2 (Table 2), were intermediate (Fig. 1A and B, strain PS36). In contrast to mutants of class 1, class 2 mutants were genetically stable under aerobic growth conditions, and the abundance of photosynthetic complexes produced under anaerobic photosynthetic conditions was lower than in mutants of class 1 yet significantly higher than in mutants of class 3. Thus, the rather broad range of mutant phenotypes suggested that residual activities of the mutant PpsR varied widely, i.e., from minor deviations from the wild-type repressor activity (class 3) to the significant decrease or apparent absence thereof (class 1).
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Repressor activities of the PpsR mutant forms in P. denitrificans. To assess the extent of impairment to PpsR repressor function, we cloned representative ppsR alleles from each mutant class into a broad-host-range vector, pRK415, and introduced these into P. denitrificans. We have previously shown that this nonphotosynthetic species, which is closely related to R. sphaeroides, is useful for studying the expression of isolated photosystem genes in the absence of components of the photosystem and the numerous regulatory factors controlling photosystem gene expression (9-11).
To monitor PpsR repressor activity, two sets of plasmids were introduced in parallel into P. denitrificans. One, pCF400
, carries the puc::lacZ
transcriptional fusion with two adjacently positioned PpsR binding
sites in the puc operon upstream region. The other
plasmid, pLX200, carries the bchF::lacZ
transcriptional fusion which contains two PpsR binding sites separated
by 125 bp.
The unrepressed level of puc::lacZ
expression in P. denitrificans, i.e., the level of
expression in the presence of the vector pRK415, was 282 times higher
than the repressed level, i.e., in the presence of the wild-type PpsR
protein expressed from plasmid pPNs (Table 2). For
bchF::lacZ expression, the unrepressed
level was 53 times higher than the repressed level (Table 2). The
mutant PpsR proteins, produced from pRK415-derived plasmids (Table 1, pPS# series), varied widely in the ability to repress
puc::lacZ and
bchF::lacZ expression in P. denitrificans. In general, the PpsR mutant repressor activities in
P. denitrificans (Table 2) correlated well with the
abundance of photosynthetic complexes and the time required for the
aerobic-to-anaerobic transition of R. sphaeroides
strains from which these ppsR alleles were isolated (Fig.
1). For example, the repressor activity of the ppsR40
mutation, cloned from a mutant of class 1, was undetectable in P. denitrificans; i.e., the ratios of the unrepressed
(ppsR absent) to repressed (mutant ppsR allele)
levels of -galactosidase were ~1 for either puc::lacZ or
bchF::lacZ expression (Table 2). The
residual repressor activity in P. denitrificans of the
ppsR36 mutation, which was cloned from a mutant of class 2, was higher than that of ppsR40, resulting in ratios of 28 and 9 for puc::lacZ and
bchF::lacZ, respectively. The residual
repressor activity of the ppsR23 mutation, which was cloned
from a mutant of class 3, was higher still, resulting in ratios of 98 and 24 for puc::lacZ and
bchF::lacZ, respectively (Table 2). The
mutant proteins repressed puc::lacZ and
bchF::lacZ in a similar, parallel
fashion; however, the extent of repression for each fusion was not
necessarily the same. The differences in the ability of a particular
PpsR mutant protein to repress puc and bchF could
be attributed to different arrangements of the PpsR binding sites;
i.e., two neighboring TGTN12ACA sites upstream of
puc versus two sites separated by ~125 bp in the case of
bchF.
As noted in a previous report (10), amino acid substitutions
K422-E (PpsR36) and C424-R (PpsR40) are located in the HTH motif of
PpsR (20) that is responsible for DNA binding (Fig. 2).
Therefore, as a first approximation one can assume that these mutations
impaired DNA binding capabilities of the altered repressor. Substitution L403-P (PpsR41) is located immediately upstream of the HTH
motif (Fig. 2), and because of the nature of a proline residue, there
is likely to be a perturbation in the secondary structure, thus
changing the context of the downstream HTH compared to the rest of the
protein. This could be the reason for low repressor activity of PpsR41
(Table 2). However, amino acid substitutions not localized to
the DNA binding domain are likely to impair the oligomeric state
of the PpsR repressor or/and its conformation. We set out to test these possibilities.
Genetic evidence for PpsR oligomerization and localization of the region of PpsR involved in oligomerization. The DNA binding site for PpsR, TGTN12ACA, consists of an inverted repeat, implying that PpsR binds as a dimer. Further, the PpsR homolog from R. capsulatus, CrtJ, has been shown to bind DNA as a dimer and possibly form a tetramer (21).
We devised a genetic test to determine whether PpsR forms oligomeric structures. We introduced both the mutant form of PpsR (expressed from a plasmid of the pPS# series) and the wild-type PpsR (expressed from plasmid pLX14P) into P. denitrificans, Pd(pLX14P), and monitored bchF::lacZ expression. We assumed that a mutant PpsR protein, which is wholly defective in oligomerization, would not interfere with functioning of the wild-type PpsR; hence, the repression level would be similar to that of the wild-type PpsR alone. However, a subset of mutant PpsR proteins which are proficient in oligomerization yet defective in repression could have a dominant negative phenotype because of the formation of mixed oligomers with the wild-type PpsR. We first tested PpsR40 (C424-R), containing a mutation in the HTH domain. Pd(pLX14P) carrying plasmid pPS40 showed an 1.9-fold-increased bchF::lacZ expression compared to Pd(pLX14P) containing vector pRK415 alone (Table 3). Hence, PpsR40 interfered with wild-type repressor presumably by forming mixed oligomers. We then tested the truncated derivative of PpsR (expressed from plasmid p[R]) that lacks the entire HTH domain. Pd(pLX14P) containing pP[R] showed an ~2.7-fold increased bchF::lacZ expression compared to Pd(pLX14P) containing vector pRK415 alone (Table 3). Hence, this truncated protein is presumed to contain the regions involved in oligomerization. The truncated PpsR protein, which consisted of the 166 carboxy-terminal residues, did not affect repression levels in Pd(pLX14P) (Table 3, plasmid p[Bg]Ns). Hence, the carboxy terminus is likely to be incapable of interacting with the wild-type PpsR. We conclude that PpsR forms dimers or higher oligomers and that the regions involved in oligomerization are localized upstream of the carboxy-terminal DNA binding region.
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The oligomeric state of native and mutant PpsR proteins.
ppsR was overexpressed in E. coli as a fusion to
GST and purified to apparent homogeneity (data not shown). Antibodies
were raised against purified PpsR protein to examine its oligomeric state in cell extracts of R. sphaeroides. Figure
3A shows a Western blot from a
nondenaturing gel probed with anti-PpsR antibody. Two cross-reacting
proteins were present in extracts from wild-type R. sphaeroides. Since one of these cross-reacting bands
disappeared in a ppsR null mutant, PPS1, we concluded that
this protein corresponded to PpsR. The nature of the second band
remains unknown. When PpsR was expressed in E. coli only,
one PpsR-specific band was observed (Fig. 3B).
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Identification of a second PAS domain in PpsR. To identify possible structural elements of PpsR involved in protein-protein interactions and conformational changes, we took a closer look at PpsR primary and secondary structures.
Previously, little sequence similarity between PpsR and other proteins in the databases could be identified, except for the carboxy-terminal HTH motif (20). Recent analysis of the PpsR protein primary structure (24) revealed that PpsR contains a PAS domain which is characteristic of a large group of prokaryotic and eukaryotic signal transduction proteins. Only limited primary sequence homology is found within the PAS domains, with highest amino acid conservation being within the PAS core and scaffold. In many instances these
domains are associated with binding-specific cofactors that lend them
signal-sensing ability. Another role of PAS domains is involvement in
protein-protein interactions. It has been shown that conformational
changes within PAS domains play a critical role in signal transduction
(reviewed in reference 28).
Closer analysis of the PpsR primary and secondary structures allowed us
to identify a second PAS domain, in addition to the one previously
suggested (24). In Figure 5 we
present an alignment of the PpsR protein, as well as its homologue from
R. capsulatus, CrtJ, with a subset of the bacterial signal
transduction proteins with identifiable PAS domains. The putative first
PAS domain of PpsR extends from P161 to T259; the second domain extends
from G280 to R367. The two PAS domains show a noticeable, albeit not very high, degree of similarity to each other, i.e., 23% identity between core boxes of the first and second PAS domains of PpsR. The
predicted secondary structure of the second PAS domain is similar to
that of the first PAS domain (Fig. 2). Both predictions fit reasonably
well with the secondary structure of PAS domains deduced from the
crystal structures of PAS domain-containing proteins, i.e., primarily
-helical PAS core, helical connector, scaffold (28).
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Construction and analysis of PpsR mutants containing deletions in the amino terminus of PpsR. Given that the carboxy-terminal region of PpsR, encompassing approximately residues 400 to 464, is presumably involved in DNA binding, and the adjacent region containing two PAS domains, from residue 161 to 367, is presumably involved in protein oligomerization and/or signal sensing, the amino-terminal region of the protein remains to be studied.
Primary structure analysis revealed that a stretch of residues from 124 to 136 is a region of low complexity, highly enriched in glutamines. This resembles the so-called Q linkers, i.e., flexible hinges connecting neighboring protein domains (29). Identification of the putative Q linker immediately upstream of the first PAS domain enabled us to suggest that the amino terminus of PpsR represents a separate protein domain. The protein sequences of PpsR and CrtJ are most divergent in the amino-terminal region, i.e., only 38% identity, compared to 47% identity in the first PAS domain and 59% identity in the second PAS domain. We have found in the databases no proteins bearing significant similarity to the amino terminus of PpsR (and CrtJ). We decided to test the significance of the amino-terminal region by constructing several deletions and testing for their effects on repressor function. Deletion of residues 2 to 10, which have no analogs in CrtJ, did not affect either PpsR repressor activity in P. denitrificans (Table 2, plasmid pPS10) or oligomeric state. However, extended amino-terminal
deletions, i.e., residues 2 to 39, or internal deletion of residues 60 to 116 resulted in marginally active and inactive repressor protein
(Table 2, plasmids pPS40 and pPS59-117, respectively). According
to our genetic test, PpsR40 did not interfere with the wild-type
repressor, suggesting a possible defect in oligomerization (Table 3),
whereas PpsR59-117 did interfere with the wild-type repressor (Table
3). However, the oligomeric state of neither of these proteins in vitro
differed from that of the wild-type PpsR. Hence, conformational changes in the mutant PpsR proteins could be invoked to explain the deleterious effect of the removal of portions of the amino terminus on PpsR repressor activity. Taken together, these results suggest that the
amino-terminal region is essential for proper tertiary structure of
PpsR, albeit its precise role is as yet unclear.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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R. sphaeroides PpsR protein functions as a key transcription factor that coordinately represses expression of photosystem genes, e.g., bch, crt, and puc, under aerobic conditions. PpsR repressor activity greatly decreases in response to anaerobiosis by as yet an unknown mechanism. Until recently, limited structural information pertaining to this protein was available. Here we attempted to gain further insight into PpsR structure and mode of action, based on analysis of the wild-type and numerous mutant proteins.
We presented genetic evidence that PpsR is likely to form oligomers. In agreement with this prediction, Ferguson plot analysis indicated that PpsR exists as a tetramer in cell extracts of E. coli. The homolog of PpsR from R. capsulatus, CrtJ, was suggested to bind its recognition site, TGTN12ACA, as a dimer. However, the presence of two recognition sites is required for efficient DNA binding by CrtJ; therefore, it was concluded that two CrtJ dimers must interact to form a tetramer (4, 21, 23). Although dimeric transcription factors are more common, a number of tetrameric DNA binding proteins have been described (1, 15, 18).
PpsR and CrtJ contain two conserved cysteine residues, C251 and C424. We considered the possibility that these cysteines could play a role in tetramer formation, e.g., by formation of intermolecular disulfide bridges. However, mutants PpsR18 (C251-R) and PpsR40 (C424-R), which contain substitutions in these conserved cysteine residues, still form tetramers. Therefore, disulfide bridges, if formed, may not be necessary for protein oligomerization.
None of the numerous ppsR mutations which led to a drastic impairment of repressor activity significantly affected the oligomeric state of the protein in the cell extracts of E. coli. This observation allows us to suggest that protein conformation within the tetramer must be critical for repressor activity. Further, it is possible that conformational changes rather than changes in the oligomeric state of PpsR represent a mechanism controlling repressor activity. The observation that PpsR contains two PAS domains strengthens this hypothesis. PAS domains are universal modules for signal sensing and transduction. In many instances these domains bind specific cofactors that respond to changes in environmental conditions. It is believed that either binding of a specific cofactor or changes in the state of a particular cofactor (e.g., changes in redox state) can significantly perturb protein conformation within a PAS domain, thus resulting in switching from the active to inactive protein conformation. Examples of redox cofactors include flavin adenine dinucleotide in the aerotaxis transducer Aer from E. coli and in the redox- and fixed nitrogen sensor NifL from Azotobacter vinelandii, heme in the oxygen-sensing histidine kinase FixL from Sinorhizobium meliloti, and 4-hydroxycinnamyl chromophore in the blue light receptor PYP from Ectothiorhodospira halophila (28).
Whether either or both of the PAS domains of PpsR is involved in cofactor binding is unknown. Neither PpsR (this work) nor CrtJ (21), purified using the E. coli overexpression systems, was found to contain cofactors. However, CrtJ showed DNA binding in vitro that was higher under oxidized compared to reduced conditions (21). One of the possibilities to explain this observation is that response to changes in redox poise could be an intrinsic property of the CrtJ and PpsR proteins. This could imply that direct oxidation-reduction of the conserved cysteine residues functions as a mechanism controlling PpsR repressor activity. One of these cysteines, C251, is present in the first PAS domain; the other, C424, is in the DNA binding region. Intriguingly, substitutions of either cysteine with arginine abolished repressor activity of PpsR (Table 2). The role of the cysteine residues requires further exploration.
Several mutations that either abolished or significantly impaired PpsR
repressor activity were localized to the PAS domains of PpsR (Table 2;
Fig. 5). N176-I (PpsR66) represents a substitution of one of the most
conserved residues in the PAS core of the first PAS domain. Residues
L248 (PpsR81) and L249 (PpsR50), located in the scaffold of the
first PAS domain, are not conserved within the PAS domains. However,
their substitutions by prolines, which are known to bring about
significant changes in protein secondary structure, could have had
major effects on the conformation of the first PAS domain resulting in
decreased repressor activity (Table 2). G288-E (PpsR77) represents a
substitution of a well-conserved residue in the PAS core of the second
PAS domain, hence the deleterious effect of the G288-E substitution on
PpsR repressor activity (Table 2). Residue C251 (PpsR18) is not well
conserved except in CrtJ; however, this position is usually occupied by
a hydrophobic residue (Fig. 5). Therefore, substitution with a charged
hydrophilic residue, C251-R, can have a dramatic effect on local
conformation. It is also possible that this residue is specifically
involved in signal sensing (see above).
Another recognized function of the PAS domains is their involvement in
protein-protein interactions. These interactions may include homologous
and/or heterologous partners. We suggest that each of the two PAS
domains of PpsR is involved in interactions with other PpsR monomers.
Surprisingly, none of the isolated mutations within the PAS domains
resulted in a significant change in the oligomeric state of PpsR in
E. coli cell extracts. This is even more striking when one
considers that the changes are located in each module of the PAS
domains, i.e., PAS core and scaffold.
The amino termini of PpsR and CrtJ are the regions least conserved
between these homologs. They show no significant similarity to known
protein sequences. The fact that extended deletions in the
amino terminus (e.g., in PpsR40 and PpsR58-117) impaired repressor activity of the mutant proteins suggests that the amino terminus plays an important role in overall protein folding. We found
that the first PAS domains in both PpsR and CrtJ are preceded by a
low-complexity region enriched in glutamines (residues 127 to 134 and
possibly 127 to 144). We suggest that the location of this region,
i.e., only several residues upstream of the first PAS domain, and its
amino acid composition make it a good candidate for a flexible hinge.
This hinge would link the PAS domains of PpsR with the amino-terminal
domain. Despite uncertainty about the role of the amino terminus, we
believe that it represents a separate protein domain with specific function.
We believe that the emerging structural organization of the PpsR and CrtJ proteins will help elucide their mode of action.
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ACKNOWLEDGMENTS |
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This work was supported by the NIH grant GM15590 to S.K., Australian Research Council grant to J.M.P. and A.G.M., and Australian Postgraduate Award to I.M.H.
We are grateful to Joyce Marshall for her contributions in the early stages of the project.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas Medical School, 6431 Fannin St., Houston, TX 77030. Phone (713) 500-5502. Fax: (713) 500-5499. E-mail: skaplan{at}utmmg.med.uth.tmc.edu.
Present address: Department of Molecular Biology, University of
Wyoming, Laramie, WY 82071.
Permanent address: Department of Biology, College of Natural
Sciences, Dong-A University, Pusan, South Korea.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, April 2000, p. 2253-2261, Vol. 182, No. 8
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