(Received for publication, October 10, 1995; and in revised form, October 30, 1995)
From the
In Rhodospirillum rubrum, CO induces the expression of
at least two transcripts that encode an enzyme system for CO oxidation.
This regulon is positively regulated by CooA, which is a member of the
cAMP receptor protein family of transcriptional regulators. The
transcriptional start site of one of the transcripts (cooFSCTJ) has been identified by primer extension. The
ability of CooA to bind to this promoter in vitro was
characterized with DNase I footprinting experiments using extracts of a
CooA-overproducing strain. CooA- and CO-dependent protection was
observed for a region with 2-fold symmetry
(5`-TGTCA-N-CGACA) that is highly similar to the consensus
core motifs recognized by cAMP receptor protein/FNR family. In vivo analysis in a heterologous background indicates that CooA is
sufficient for CO-dependent expression, implicating it as the likely CO
sensor.
Exposure of the purple nonsulfur bacterium Rhodospirillum
rubrum to CO stimulates the expression of the coo regulon, which consists of at least two transcriptional units.
Among the products of this regulon are a carbon monoxide dehydrogenase
(CooS), an Fe-S protein (CooF), and a hydrogenase (CooH), where the two
former proteins have been purified and
characterized(1, 2, 3, 4, 5, 6) .
This CO-oxidizing system functions under anaerobic conditions to
oxidize CO to CO, allowing growth on CO as sole energy
source(7) . The cooFSCTJ region has been cloned,
sequenced, and mutationally characterized, verifying the requirement
for the encoded products for oxidation of CO(7, 8) . (
)
The mutational analysis has also indicated that cooFSCTJ is organized in a single transcriptional unit.cooH lies at the 3` terminus of the other known
CO-regulated transcript, but this transcript has not yet been fully
sequenced at the 5` end. cooH is located 5` of cooF and is separated from it by 450 nucleotides of noncoding DNA (Fig. 1B).
Figure 1:
Identification of the transcriptional
start site for cooF. Panel A shows the result of
primer extension of the region upstream of cooF. A 22-mer
oligonucleotide (Primer 2), which is complementary to the coding strand
in region -154 to -133 relative to the translational start
site of cooF was 5`-end-labeled and used to prime the reverse
transcriptase reaction. The sequencing ladder (G, A, T, C) used the
same primer but was not end-labeled. +CO refers to RNA
extracted from R. rubrum cells induced with CO, while the
-CO lane refers to RNA from uninduced cells. Asterisks indicate the 5`-end of the major and minor RNA
species detected. The fact that the primer for the reverse
transcription was end-labeled and the one for sequencing was not caused
a one-base shift in reading the transcriptional start site. Panel B provides a schematic of the region upstream of cooF. The
space between the cooH and cooF genes is 450 bp. The
transcriptional start site predicted by the major RNA species from panel A is indicated, as is the region protected in the
footprinting experiments shown in Fig. 2A. Boldface letters in the CooA target region represent a 2-fold symmetric
sequence that is highly similar to the consensus sequence motif
recognized by CRP/FNR (Fig. 2B). The underlined CG and GG residues at -13, -14, -25, and
-26 are characteristic of -dependent
promoters.
Figure 2:
Identification of the CooA binding site. Panel A shows the result of a DNase I footprinting experiment.
A 294-bp EcoRV-EagI fragment containing the promoter
region of cooF was used in this assay. CooA refers to extracts of UR407 (cooA::aacC1) and CooA
refers
to extracts of UR459 (the CooA-overproducer with cooA under
PnifH control). The numbers reflect micrograms of protein in
each assay, and the + and - on the line labeled CO reflect the presence or absence of CO in the binding reaction; all
experiments were performed anoxically. The G+A lane
represents the Maxam-Gilbert sequencing marker. The box on the
right side indicates the region protected in this experiment. Panel
B shows a comparison of the CRP and FNR consensus binding sites
with the detected CooA-binding site.
Our previous mutational studies revealed
that CooA, ()which is apparently encoded on it own
transcript on the 3` side of cooFSCTJ, is essential for the
expression of the coo regulon of R. rubrum in
response to CO(9) . The sequence of CooA predicts that it is a
member of the CRP/FNR family of transcriptional regulators(9) ,
with a putative DNA-binding domain that is highly similar to that found
in CRP and FNR. Modeling the sequence of CooA on the known CRP crystal
structure (10) predicts the presence of four Cys and one His
residues adjacent to the region known to bind cAMP in CRP(9) .
These residues suggest the possibility that CooA contains a metal
center at this position, which might be expected if CooA binds CO. A
particularly interesting question in this area is how the binding of a
molecule as small as CO might induce a similar conformation change in
CooA as that caused by cAMP binding in CRP.
To test the model of CooA as a CO-binding transcriptional activator, we have sought evidence for CO- and CooA-dependent DNA binding. The results described herein support the above model, and the assay of DNA-binding activity of CooA will aid in the purification of CooA for more direct analysis.
For RNA isolation, cultures to be CO-induced were grown photoheterotrophically to an optical density of 1 at 680 nm, whereupon CO was added to a final concentration of 30%; uninduced culture received no additions. The cultures were agitated under illumination (8) for 6 h.
The CooA-overexpressing strain (UR459) was
grown under nif-derepression conditions in malate-glutamate
medium (11) to an OD of 2.0, and the expression
of nifH promoter was monitored by nitrogenase activity (12) .
The P and nifH coding material remaining in pDWS126 (77 bp total) was deleted to
create a junction between the ribosome binding site of nifH and the initiation codon of cooA in the following way. A
primer,
5`-CGATGTTGAAACGAGGCGGCATGGAATCAATCCTTTTCTTCGGTGATCCGGTCTTAAGGCGGG,
(double underline indicates a base change (C to T) from the wild-type
P
region to create a new AflII site (single
underline)) was synthesized (Genosys Biotechnologies) and used for
site-directed mutagenesis by a modification of the unique site
elimination procedure(21) , utilizing a single primer
incorporating the desired deletion as well as a selectable restriction
site (Bsu36I) loss. The desired plasmid (pDWS131) was
identified by the presence of an new site in the plasmid (AflII) derived from the primer. The P
region
and cooA on pDWS131 were verified by sequencing in one
direction. pDWS131, which is a mobilizeable plasmid that does not
replicate in R. rubrum, was used to transform Escherichia coli strain S17-1(17) , and the
resulting strain was mated with R. rubrum strain UR2 (coo
).
Strains with pDWS131
integrated into the UR2 chromosome by homologous recombination were
selected for kanamycin (15 µg/ml). A single transconjugant, R.
rubrum strain UR459, was used for further study.
In order to more precisely identify the 5`-end of the cooF mRNA, a second primer (Primer 2) was designed that hybridized about 150 nucleotides upstream of cooF coding region. Results with this primer showed that the major transcript from the cooF promoter initiates with the A nucleotide positioned 257 bp upstream from the start codon of cooF. A minor product starting six nucleotides upstream of that site was also observed. These primer extension products were only detectable in the CO-induced culture (Fig. 1A), indicating that the effect of CO is on the accumulation of coo mRNA. Fig. 1B shows a schematic of the transcription start site relative to other features in the region, including the putative CooA-binding site (see below).
Extracts of UR459
(P::cooA) were examined by SDS-polyacrylamide
gel for the presence of a protein band corresponding to CooA. The
extracts of UR2 and UR407 (cooA::aacC1)(9) ,
grown under the same conditions as UR459, were used as controls, as
CooA was deficient in UR407 and is not expected to be detectable in
UR2. A band migrating at about 25 kDa, the predicted molecular mass of
CooA, was significantly more intense in extracts of UR459 than in those
of UR2 or UR407 (data not shown).
In DNase I protection experiments (Fig. 2A), a specific pattern of protection was detected only with extracts from the CooA-overproducing strain in the presence of CO. No protection was observed in the same extract in the absence of CO, nor in the extract of the cooA mutant (UR407) regardless of the presence of CO in the binding reaction. This protection was not detectable with extracts of wild-type in presence of CO (data not shown), presumably due to low levels of CooA.
The site protected by CooA covers 28 bases (position -27 to -54) and contains a sequence of 2-fold symmetry (Fig. 1B) that is highly similar to the consensus sequence motif recognized by CRP/FNR in E. coli (Fig. 2B).
Gel retardation analysis with the same DNA fragment and extracts also revealed a DNA-protein complex whose presence requires both CooA and CO (data not shown). This CooA- and CO-dependent complex is very large; it remained in the wells of a 5% polyacrylamide (19:1 acrylamide/bis ratio) gel but entered a 5% polyacrylamide (37.5:1 acrylamide/bis ratio) gel, suggesting the presence of additional proteins in this complex.
Our previous mutational studies and sequence analysis of cooA led us to predict that CooA is a CO-sensing
transcriptional activator similar to CRP and FNR. The work presented in
this paper and elsewhere strongly supports the hypothesis that CooA is
a CO-sensing protein responsible for controlled expression of the coo region in a fashion reminiscent of the action of CRP: (i)
Northern blot analysis and primer extension experiments
demonstrate that CO affects mRNA accumulation; (ii) CooA is sufficient
for CO-dependent expression in R. sphaeroides; (iii) DNA
binding appears to be CooA- and CO-dependent in vitro; (iv)
the detected CooA target site is very similar to the CRP/FNR consensus
binding site; and (v) CooA is very similar to CPR and FNR in the
helix-turn-helix DNA binding domain(9) .
We initially looked
for the CooA-binding site by both footprinting and gel retardation
assays in the 250-bp EagI-BsmI region immediately
upstream of cooF (Fig. 1B), in part because a
strain (UR284) with an insertion at the EagI site (Fig. 1B) displayed CooS activity in a CO-dependent
manner(8) . We now believe that the observed expression in
UR284 reflects transcription from P through the
Kan
insertion; the Kan
gene is derived from
pUC4K and apparently lacks transcriptional terminators. No CooA- or
CO-dependent DNA binding could be found between the EagI site
and cooF (data not shown), however, and the transcriptional
start site identified in this paper is clearly the physiologically
significant one in vivo.
While the detected transcription
start lacks the -10 and -35 sequences expected at a typical E. coli promoter, a typical E. coli
recognition sequence is present, with GC and
GG at -13 and -25, respectively (25) (Fig. 1B). An interaction of CooA with
would be interesting as no CRP- or FNR-controlled
promoters are known to be recognized by
.
The center of the two-fold symmetry of the CooA binding site is at -43.5 with respect to the transcriptional start point of cooF. This distance is similar to the location of the CRP sites in class II CRP-dependent promoters (e.g. galP 1) and FNR sites in the FNR-dependent promoters(26, 27) . In CRP, the specific interaction between the side chains of the protein and a given base within the core motif 5`-TGTGA-3` have been reviewed(28, 29) : Arg-180 and Glu-181 directly contact the 5`-G and the nucleotide of complementary to the 3`-G, respectively. It is possible that Arg-177 of CooA, which is in the homologous position of Arg-180 of CRP, contacts the 5`-G of the CooA target site 5`-TGTCA-3`. The absence of a Glu in CooA corresponding to Glu-181 in CRP is consistent with the fact that there is a 3`-C instead of a 3`-G in the CooA target site (Fig. 2B).
We are currently employing the described gel shift as a functional assay for the purification of CooA. Analysis of the purified protein, together with the eventual determination of the CooA-regulated promoter upstream of cooH, will significantly increase our understanding of this regulatory response.