(Received for publication, April 10, 1997, and in revised form, May 9, 1997)
From the § Biochemistry Program, Department of Chemistry, and the ¶ Department of Biology, Indiana University, Bloomington, Indiana 47405
Previous genetic analysis indicated that the
photosynthesis gene cluster from Rhodobacter capsulatus
coded for the transcription factor, CrtJ, that is responsible for
aerobic repression of bacteriochlorophyll, carotenoid, and light
harvesting-II gene expression. In this study, we have heterologously
overexpressed and purified CrtJ to homogeneity and shown by gel
mobility shift assays that CrtJ is biologically active. DNase I
footprint analysis confirms molecular genetic studies by showing that
CrtJ binds to conserved palindromic sequences that overlap the 10 and
35 promoter regions of the bchC operon. Graphs of the
percentage of DNA bound versus protein concentration show
sigmoidal curves, which is highly indicative of cooperative binding of
CrtJ to the two palindromic sites. A binding constant for interaction
of CrtJ with the palindrome that spans the
10 region was calculated
to be 4.8 × 10
9 M, whereas affinity for
the palindrome that spans the
35 region was found to be 2.9 × 10
9 M. Binding of CrtJ to the bchC
promoter region was also found to be redox-sensitive, with CrtJ
exhibiting a 4.5-fold higher binding affinity under oxidizing
versus reducing conditions.
Rhodobacter capsulatus is a purple nonsulfur photosynthetic bacterium that regulates synthesis of its photosynthetic apparatus in response to alterations in oxygen tension and light intensity. Oxygen tension at atmospheric levels (21%) results in virtual suppression of photopigment production, whereas reduced oxygen tension (<1%) promotes cellular differentiation that results in the formation of an intracellular membrane that houses the light-driven energy-generating photosystem (1). Oxygen regulation of photosystem synthesis is known to be controlled, in part, by regulating transcription of photosynthesis genes (reviewed in Refs. 2 and 3).
Regulation of photosynthesis gene expression by oxygen tension is known to be controlled by many different transcription factors (reviewed in Refs. 2 and 3). One regulatory circuit, RegB-RegA, is a phosphorylation cascade responsible for anaerobic activation of light harvesting and reaction center structural genes that are coded by the puf, puh, and puc operons. RegB is a histidine kinase that initiates a phosphorylation cascade when the cells are growing in an anaerobic environment (4-6). In a previous study, we observed that disruption of an open reading frame termed crtJ (ORF469) resulted in the aerobic production of photopigments (7). Genetic and transcriptional studies have indicated that crtJ appears to code for an aerobic repressor of bacteriochlorophyll (bch), carotenoid (crt), and light harvesting-II (puc) gene expression (8-12). We also observed that cell-free extracts derived from wild type R. capsulatus cells are able to promote the formation of a stable gel mobility shift with a bchC promoter fragment, whereas extracts from a crtJ-disrupted strain were unable to do so (11). Gel shift (11) and mutational analysis (12), have indicated that CrtJ is most likely interacting with a conserved palindrome (gTGT-N12-ACAc) that is present in the CrtJ-regulated bch, crt, and puc promoters. However, direct proof that CrtJ recognizes the conserved palindrome sequence has not been demonstrated.
In this study, we have heterologously overexpressed and purified CrtJ to homogeneity. Gel mobility and DNase I protection analysis were utilized to demonstrate that CrtJ indeed binds to a conserved palindrome sequence that is present in two copies of the bchC promoter. We also demonstrate that binding of CrtJ to the bchC promoter is affected by the redox state of the binding buffer.
Strain NM522 was used for routine cloning procedures. For overexpression of CrtJ, strain BL21(DE3) was utilized (13). Plasmids pRPS404 (14), pUC19 (15), pT7-7 (16), and pET28(a)+ (16-18) have been described previously. Luria broth was used for agar solidified plates and for liquid cultures (19). Ampicillin and kanamycin were used at 100 and 30 µg/ml, respectively.
Construction of a CrtJ Overexpression VectorCrtJ was
expressed in Escherichia coli using a T7 RNA
polymerase-based overexpression system that appended the amino terminus of CrtJ with a His6 tag (Novagen). For this construction,
the CrtJ coding region (14,821-16,209 base pairs of the photosynthesis gene cluster; Ref. 20) was amplified using a polymerase chain reaction
(PCR).1 The upstream and downstream
oligonucleotide primers, 5CCCATATGCGACGGGAGGCCTTGCA and
5
CCTCTAGAACGGTCCTGGAGCAGGCGTT, respectively, were
designed to contain an NdeI restriction site at the
crtJ start codon and an XbaI site at the stop
codon (underlined bases). The PCR-amplified fragment was subsequently
cloned into SmaI-digested pUC19 and then subcloned into the
NdeI-HindIII restriction sites of pET28(a)(+) (Novagen), resulting in the recombinant plasmid,
pET28::CrtJ.
The expression
plasmid pET28::CrtJ was transformed into the pT7 RNA
polymerase expression strain BL21(DE3), and CrtJ was then expressed to
high levels by isopropyl--D-thiogalactopyranoside (IPTG)
induction as described previously (16, 17). The cell pellet from a
100-ml culture was then resuspended in 4 ml of ice-cold binding buffer
composed of 20 mM Tris-HCl, pH 7.9, 5 mM
imidazole, 0.5 M NaCl, 0.1% Nonidet P-40, and the cells
were then disrupted by sonication. The lysate was then clarified by
centrifugation at 39,000 × g for 20 min with the
supernatant further clarified by filtration through a 0.45-µm
membrane (Gelman Sciences) to prevent clogging of the resin during
column chromatography. Purification of His6-appended CrtJ
was performed using nickel column affinity chromatography as described
by the resin manufacture (Novagen). Fractions containing the highest
CrtJ concentrations as visualized by SDS-polyacrylamide gel
electrophoresis were pooled, yielding a typical concentration of 0.30 mg/ml and dialyzed overnight in 1 liter of buffer composed of 50 mM Tris-HCl, pH 7.9, 50 mM potassium acetate, 1 mM EDTA, and 20% glycerol. The purified protein was then
partitioned into 30-µl aliquots, subjected to rapid freezing using a
dry ice/ethanol mixture, and stored at
70 °C.
PCR amplification and purification of a 32P-labeled DNA probe containing the bchC promoter region was prepared as described by Ponnampalam et al. (11). For determination if purified CrtJ was active, a gel mobility shift assay was performed that compared DNA binding properties of purified CrtJ with that of a crude lysate that was prepared as described by Ponnampalam et al. (11). For this analysis, 3 µl of 32P-end-labeled bchC DNA probe (10 fmol/3 µl) was added to 2.5 µl of poly(dI·dC) nonspecific competitor DNA (0.20 µg/µl). The DNA solution was then added to a 15-µl reaction containing either purified CrtJ protein (0.50-1.0 µg) or crude lysate (2 µg) in a reaction buffer composed of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.1, 50 mM potassium acetate, 20% glycerol (v/v). Each reaction was then incubated for 20 min at 30 °C, loaded on a native 6% Tris-glycine-EDTA-buffered polyacrylamide gel, and electrophoresed at 16 mA for 2.5 h at 4 °C.
To ascertain if DNA binding of purified CrtJ is redox-sensitive,
purified CrtJ was diluted into a 30-µl reaction buffer containing 9 fmol of 32P-end-labeled bchC DNA probe and 3 µl of a 10 mg/ml heparin solution. To facilitate the transport of
electrons from the oxidizing/reducing agent to the protein center, 1.5 µl of ethanol and 1.0 µl of water-soluble redox mediators were also
added to the mix (21). The ethanol-soluble mediators used were
1,4-benzoquinone (2.5 µg/ml),
N,N,N,N
-tetramethylphenylenediamine (3.0 µg/ml),
diaminodurene (12 µg/ml), 1,2-naphthoquinone (3.0 µg/ml),
5-hydroxy-1,4-naphthoquinone (3.0 µg/ml), duroquinone (4.0 µg/ml),
2,5-dihydroxy-1,4-benzoquinone (3.5 µg/ml), anthraquinone (saturated), and 2-hydroxy-1,4-naphthoquinone (3.0 µg/ml). The water-soluble mediators used were phenazine methosulfate (7.0 µg/ml),
anthraquinone-2,6-disulfonate (7.5 µg/ml), anthraquinone-2-sulfonate (7.5 µg/ml), benzyl viologen (1.0 µg/ml), and methyl viologen (1.0 µg/ml). The protein samples were incubated for 30 min on ice in the
presence of 10 mM sodium dithionite, argon gas, oxygen gas,
or 20 mM potassium ferricyanide. Oxygen gas was bubbled
through the sample continuously for 5 min. Argon gas was bubbled
through the protein sample alternately with degassing by vacuum several times prior to addition of argon. After the 30-min redox incubation period, 3 µl of 32P-end-labeled probe (10 fmol) was added
to the samples, which were then incubated for an additional 20 min at
30 °C. 3 µl of heparin (10 µg/ml) was then added as a
competitor, and the samples were then electrophoresed at 4 °C as
described above.
A 183-base pair DNA segment containing
the bchCXYZ promoter region was obtained by PCR
amplification using oligonucleotide primers 5-GTTCGGACCCGGCTTTGACC and
5
-TTCACCAAGGTGTCGAAACCG with amplification conditions as described for
the gel mobility shift assays. For selective labeling of DNA strands,
one of the primers in the PCR reaction was
5
-32P-end-labeled prior to amplification. The amplified
segment was purified as described for the gel mobility shift assay.
Binding of purified CrtJ to the DNA fragment prior to DNase I digestion involved conditions similar to those described for the gel shift assays
with the reaction mix composition containing 4 µl of end-labeled DNA
(12 fmol), 82 µl of gel mobility shift buffer, and 9 µl of CrtJ at
varying concentrations. After incubating the binding reaction at
30 °C for 20 min, DNase I (0.50 µg/ml) was added to the mixture, and the digestion was allowed to proceed for 2 min at room temperature. The reaction was then stopped by the addition of 100 µl of DNase I
stop solution and electrophoresed on a 5% urea-denaturing Long Ranger
polyacrylamide gel as described previously (22). A modified Maxam and
Gilbert G + A chemical sequencing reaction was used for determining the
location of DNase I protection (23).
EC50 values for CrtJ binding to the bchC promoter region were calculated using a modified method of Brenowitz et al. (24). Briefly, CrtJ DNA binding isotherms to the bchC promoter region were generated by DNase I footprint titration assays using 2-fold protein dilutions ranging from 4 ng to 4 µg. The gel was then analyzed for CrtJ binding isotherms using a PhosphorImager (Molecular Dynamics) to quantitate the level of CrtJ protection of a single band in the upstream and downstream palindromes. Values were corrected for loading by normalization with a band from an unprotected region.
Nitrocellulose filter binding assays according to the method of Witherell and Uhlenbeck (25), were utilized to determine the fraction of isolated CrtJ that was active in DNA binding, the level of which was then used to correct the EC50 values for CrtJ binding to the bchC promoter region. For this determination, 1.2 pmol (62.5 ng) of purified CrtJ was incubated with varying amounts (from 0.04 to 3.65 pmol) of 32P-labeled bchC DNA probe that was identical to the probe used for the DNase I footprint titration assay. The sample volume used for each reaction was 30 µl consisting of 10 µl of DNA sample (of varying dilutions), 5 µl of heparin (100 µg/ml), 9 µl of CrtJ (1.2 pmol), and 6 µl of binding buffer. The reactions were incubated at 30 °C for 10 min and then filtered through 0.45-µm pore size, pure nitrocellulose filters (number 9138/2, Schleicher and Schuell), which had been previously soaked in binding buffer (50 mM Tris-HCl, pH 8.0, 50 mM potassium acetate, and 20% glycerol) for 2 h at room temperature. The filtration time was relatively rapid, with most of the filtrate passing through the nitrocellulose filter within 60 s. The filters were then air-dried for 30 min and counted in a Packard scintillation counter (model Tri-Carb 2100TR) using Bio-Safe II Biodegradable Counting Mixture (Research Products International Corporation). Retention efficiencies for the 32P-labeled DNA calculated as the number of counts retained on the nitrocellulose filter compared with the number of counts retained on the filter at saturating levels of the protein ranged from 60 to 95%. A control assay was also done in which no protein was added to the varying amounts of DNA used to ascertain the background level of DNA that bound to the nitrocellulose filter.
Hill Coefficient DeterminationThe Hill coefficient was
determined by using a nonlinear regression analysis to fit the Hill
equation given by y = a/1 + (x/E)n, where a is the
maximum value of y as x approaches infinity, y corresponds to the fractional occupancy of the binding
site on the DNA molecule, x is the protein concentration
(nM), E is the EC50 value, which
corresponds to half-maximal saturation of each binding site, and
n is the Hill coefficient. The Hill coefficients for the
binding of CrtJ to both the upstream and downstream palindromes were
obtained from a DNase I footprint titration assay from which site
binding isotherms were generated. The Hill coefficients were then
determined from a graph of fractional saturation versus
protein concentration.
A His-tagged
version of CrtJ was overexpressed in E. coli using a
T7-based overexpression system. As demonstrated by the
SDS-polyacrylamide gel electrophoresis polypeptide profile in Fig.
1, IPTG-based induction of T7 RNA polymerase resulted in
high level overexpression of CrtJ, which was predominantly found in the
soluble fraction (lane 3). CrtJ was subsequently isolated to
a high level of purity (>95% as judged by Coomassie Blue staining;
lane 4) by affinity binding of the His6-tagged
CrtJ to a Ni2+ column.
Gel retardation (mobility shift) assays were subsequently performed to
ascertain if isolated CrtJ was active. As a control, we performed a gel
retardation assay with crude cell lysates obtained from wild type and
CrtJ-disrupted R. capsulatus cells. In confirmation of
previous results (11), we observed that a DNA fragment that contains
the bchC promoter region was shifted to a reduced
electrophoretic mobility when incubated with the wild type extract
(Fig. 2, lane 2). This mobility shift is
absent in extracts derived from the crtJ-disrupted strain
DB469 (lane 3), suggesting that the mobility shift observed
with the wild type extract resulted from an interaction of CrtJ with
the bchC promoter region. This supposition is supported by
mobility shifts obtained with increasing concentrations of purified
CrtJ (lanes 4-6) that has an electrophoretic mobility identical to that observed with wild type crude extracts. These results
indicate that heterologously expressed CrtJ was isolated in a properly
folded form and that it has specificity for a sequence within the
bchC promoter region.
CrtJ Binds to a Duplicated Palindrome in the bchC Promoter Region
DNase I protection (footprint) analysis of CrtJ binding to
the bchC promoter region was performed on both DNA strands
by selectively 5-end labeling either the top or bottom strand. As seen
from the DNase I digestion patterns in Fig. 3, CrtJ
protects a region of the top strand ranging from
3 to
52 and on the
bottom strand from
1 to
49. Inspection of the protected DNA
sequence (Fig. 3) indicates that this region contains two copies of the
palindromic sequence TGT-N4-TCAA-N3-ACA, one of
which flanks the
10 and the other the
35 region of the
bchC promoter (11, 12, 24). Several sites hypersensitive to
DNase I digestion are also observed on the top and bottom strands
(Figs. 3 and 4). These hypersensitive strands extend 36 base pairs upstream and 58 base pairs downstream from the region of
CrtJ protection.
Determination of Percentage of Active CrtJ
The fraction of
CrtJ that is active in DNA binding was determined according to the
filter binding method of Witherell and Uhlenbeck (25), which involves
quantitating the amount of maximal DNA probe bound with a known amount
of protein. If the oligomerization state of the protein bound to the
DNA probe is known, then it is possible to calculate the percentage of
fraction of protein that is active. For our analysis, the CrtJ protein
concentration was held constant at 1.2 pM (at approximately
half saturation) with the 32P-labeled bchC DNA
probe varied in the vicinity of the protein (CrtJ) concentration. As
shown in the DNA titration curve in Fig. 5, the amount
of DNA bound by the filter rises to a saturation point of 0.11 pmol of
DNA (note that this value has been corrected for the efficiency of
probe retention by the filter as described under "Materials and
Methods"). Assuming that the DNA probe maximally binds to the filter
when both palindromes are fully occupied and that two CrtJ bind per
palindrome for a total of four CrtJ/probe, then at saturating levels of
input DNA bound (0.11 pmol) there must be 0.44 pmol of protein bound by
the filter. Thus, the fraction of protein that is active corresponds to
0.44 pmol of protein retained by the filter/1.2 pmol of input protein
in the assay, which corresponds to an active protein fraction of
36.7%.
EC50 Values and Hill Coefficient Determination of CrtJ Binding to the Upstream and Downstream Palindromes of the bchC Promoter Region
Binding isotherms for interactions of CrtJ to each of the
individual palindromes were determined using a DNase I footprint titration assay according to the method of Brenowitz et al.
(24). This method involves quantitating, by PhosphorImager analysis, CrtJ-mediated protection of a DNase I digestion site in each of the
palindromes using small increments of CrtJ and correcting for
background. The optical density ratios are then converted to fractional
protection values. However, because even at saturating levels of
protein, the DNA is not completely protected from DNase I digestion,
the fractional protection values have to be converted to fractional
saturation values. The fractional saturation values are then plotted
against the logarithm of the protein concentration (Fig.
6). As shown in Fig. 6, CrtJ protection of the upstream (21 to
52) and downstream (
1 to
20) palindromes exhibits
sigmoidal curves of protection with an average Hill coefficient of 2.5 for each binding site. Since the Hill coefficient is greater than 2, it
indicates that there are cooperative interactions among three or more
protein subunits that are binding to the two palindromic binding sites.
This further supports the hypothesis that CrtJ binds in a multimeric
form, possibly as a dimer to each palindromic site and that
tetramerization of these dimers induces a cooperative interaction (see
"Discussion"). From the fractional saturation values, we can also
determine an EC50 value for binding of CrtJ to the upstream
and downstream palindromes. The values obtained, after correcting for
37% active protein, are values of 2.9 × 10
9
M for binding to the upstream palindrome and 4.8 × 10
9 M for binding to the downstream
palindrome.
CrtJ Binding to the bchC Promoter Region Is Redox-sensitive
Since previous genetic analysis indicated that
CrtJ most likely functions as an aerobic repressor (11), we next
addressed whether binding of CrtJ to the bchC promoter was
affected by alterations in the redox state of the binding buffer. For
this analysis, we first preincubated CrtJ for 30 min in the presence of
a binding buffer that contained soluble redox mediators (to facilitate
the transport of electrons from the oxidizing/reducing agent to the protein center) (22) as well as various oxidizing/reducing agents. After preincubation, a 32P-end-labeled bchC DNA
probe was added to the samples, incubated for an additional 20 min, and
then subjected to gel electrophoresis. As shown in the gel shift assays
in Fig. 7, preincubation of CrtJ in the presence of a
binding buffer that was saturated with oxygen (by bubbling molecular
oxygen into the binding buffer) or 20 mM potassium
ferricyanide, which have redox values of approximately +800 and +450
mV, respectively, promoted excellent binding conditions. In contrast,
preincubation of CrtJ under conditions where oxygen was replaced with
the redox neutral gas argon or with 10 mM of the strong
reducing reagent sodium dithionite (approximately 600 mV) resulted in
a significant reduction of binding activity.
Three separate reactions were performed to determine if the observed
redox response was reversible. In the first reaction, CrtJ was
incubated separately with either 10 mM sodium dithionite or
20 mM potassium ferricyanide for 1 h. In a second
reaction, CrtJ was first incubated for half an hour with 10 mM sodium dithionite, an excess of potassium ferricyanide
(20 mM) was added, and then the reaction was incubated for
a further 30 min. As shown in Fig. 8, the inhibitory
effect of reducing conditions is indeed reversible, since the addition
of the excess potassium ferricyanide to the CrtJ sample that had been
inhibited with dithionite promoted the formation of a stable
DNA-protein complex in the gel mobility shift assay (lanes 8 and 9). Thus, CrtJ appears to be capable of sensing
environmental redox values and adjusting binding accordingly.
We next performed gel mobility shift assays with small increments
(0.05-0.10 µg) of CrtJ to generate binding isotherms for the
determination of EC50 values (effective concentration of
CrtJ for 50% response) of CrtJ binding to the bchC promoter
region under varying redox conditions. Cooperative binding of CrtJ to the two palindromic sites of the bchC promoter region is
also revealed when the percentage of DNA bound is plotted against
protein concentration. Sigmoidal curves were obtained indicating
cooperativity between the two binding sites (Fig. 9).
The EC50 value for CrtJ binding to the bchC
promoter region indicates that CrtJ binding affinity for the
bchC promoter increases 4.5-fold under oxidizing versus reducing conditions (Table I). The
corrected EC50 values for binding of CrtJ to the
bchC promoter region (Table I), as based on gel mobility
shifts (the values of which were corrected for percentage of active
fraction and for the binding of four CrtJ/promoter fragment), indicates
that binding to the promoter fragment is 6.0 × 109
M under oxidizing conditions (oxygen) and 2.7 × 10
8 M under reducing conditions (sodium
dithionite). Intermediate EC50 values were obtained for
less severe oxidizing/reducing conditions. These values are 8.4 × 10
9 M and 1.9 × 10
8
M for potassium ferricyanide and argon, respectively (Table
I).
|
Our results demonstrate that highly purified His-tagged CrtJ can
be readily isolated in an active form by a one-step purification using
a Ni2+ charged column. The isolated protein is active as
evidenced from gel mobility shift assays where purified CrtJ shows an
identical shift with that of the crude lysate of wild type R. capsulatus cells and by the results of our DNase I footprint
titration assays. The protection pattern of CrtJ binding to both the
top and bottom strands of the bchC promoter region shows an
area of protection extending approximately 52 base pairs from 1 to
51. This region contains two conserved palindromic sequences
TGT-N12-ACA that are centered around the
70-like promoter sequences present at the
35 and
10
regions of the bchC operon (11, 12, 26). Recognition of this
palindrome by CrtJ is also supported by prior mutational analysis,
which indicated that they were involved in the binding of an aerobic repressor (12).
Evidence at hand indicates that cooperative interactions occur between
CrtJ bound at the two palindromes. Cooperativity is indicated by the
formation of sigmoidal curves and by the Hill coefficient obtained for
CrtJ binding to the two palindromic sites, which indicates that this
interaction involves more than 2.5 CrtJ polypeptides. We also have
unpublished data that indicates that CrtJ binds very poorly to DNA
segments that contain only one of the two identified bchC
palindromes.2 Given well characterized
interactions observed among other prokaryotic transcription factors
such as the cI repressor, the nitrogen regulator NtrC,
and the lacI repressor in E. coli (27-29), the most likely scenario is that CrtJ binds as a dimer to each of the
palindromes and that the dimers cooperatively interact to form stable
tetramers.
The EC50 values of CrtJ binding to the bchC
promoter region under oxidizing conditions is 6.0 × 109 M, as based on gel mobility shift
results, which is the same order of magnitude as that of the
cI repressor binding to the right operator site,
OR1 (under "physiological conditions", cI repressor has a Kd of 3 × 10
9
M for OR1 (13, 30, 31)). This value is,
however, several orders of magnitude lower than that observed for other
repressors, such as the lac repressor, which has a binding
affinity of 10
12 M (32). The modest affinity
of CrtJ binding to the bchC promoter may reflect the fact
that CrtJ-regulated promoters are only moderately repressed
(1.5-2-fold) by CrtJ (11, 12), which is contrasted by the 1,000-fold
(32) repression exhibited by the lac repressor.
Since previous genetic analysis indicated that CrtJ functions as an aerobic repressor (8-12), it was perhaps not surprising to find that CrtJ binds to the bchC DNA fragment better under oxidizing versus reducing conditions. The calculated EC50 value of CrtJ binding to the bchC promoter region under oxidizing conditions is 4.5-fold higher than that observed under reducing conditions, suggesting that CrtJ may have redox sensing capabilities. The effect of altering the redox state on CrtJ binding is reversible, suggesting that reducing conditions are not nonspecifically affecting DNA binding activity. Furthermore, CrtJ binding is best under highly oxidizing conditions (such as oxygen-saturated binding buffer), which is a condition that inhibits binding of other redox-responding DNA binding proteins such as FNR, DtxR, SoxR, and Fur. These latter redox-responding proteins have oxygen-labile iron or iron-sulfur clusters that are required for optimal DNA binding capabilities (32-40). Although the redox-sensing component within CrtJ is yet to be elucidated, we feel that it is unlikely to have a redox-responding iron center. Inspection of the CrtJ sequence indicates no obvious Cys-rich iron binding motifs such as that found in iron binding transcription factors. Atomic absorption spectrometry and EPR of isolated CrtJ also shows no significant levels of iron in the isolated protein preparations.2 A recent study by Gomelsky and Kaplan (41) indicates that in Rhodobacter sphaeroides a second protein (AppA) may also be involved in controlling redox-sensitive binding by CrtJ. Although no experimental evidence has been obtained for a function for AppA in controlling CrtJ activity, it is possible that AppA may be sensing redox and somehow transmitting this information to CrtJ. A homolog for AppA in R. capsulatus has not yet been described, so at this time we are unable to biochemically ascertain whether AppA affects the in vitro redox response of CrtJ DNA binding.
Finally, there are several studies that indicate that CrtJ may be a global repressor of pigment biosynthesis genes and that its presence is conserved in diverse species of anoxygenic photosynthetic bacteria. For example, sequence and genetic analysis of the R. capsulatus photosynthesis gene cluster indicates the presence of similar palindromic motifs within putative promoter sequences for bacteriochlorophyll, carotenoid, and light harvesting-II structural genes (2, 12, 20, 26, 42, 43). Sequence analysis of the puc promoter from such diverse species as R. sphaeroides (44), R. capsulatus (45), and Rhodopseudomonas palustris (46) also shows the presence of the palindrome that is characterized in this study, indicating that CrtJ may be conserved among a diverse group of anoxygenic photosynthetic bacteria. Thus, any insights into the mechanism of redox responsiveness of CrtJ binding to photosystem promoters could also provide insight into how these additional organisms control photosystem development.
We thank Dr. John Richardson for helpful comments regarding this study.