1 Department of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944, USA
2 Department of Microbiology and Parasitology, University of Queensland, Brisbane 4072, Australia
3 Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030, USA
Correspondence
Mark Gomelsky
gomelsky{at}uwyo.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is L19596.
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INTRODUCTION |
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Oxygen is a critical environmental signal that regulates formation of the PS in R. sphaeroides. In the presence of oxygen, formation of the PS is inhibited. A decrease in oxygen tension stimulates expression of the PS genes and subsequent synthesis of PS components and formation of a functional PS. Several regulatory factors controlling PS gene expression in response to oxygen have been identified (reviewed by Pemberton et al., 1998; Zeilstra-Ryalls et al., 1998
; Gregor & Klug, 1999
; Oh & Kaplan, 2001
). Under conditions of low or no oxygen, light intensity and quality affects the abundance of photosynthetic complexes and PS gene expression. Much less is understood about light-dependent regulation of PS formation in R. sphaeroides (Zeilstra-Ryalls et al., 1998
; Braatsch et al., 2002
; Masuda & Bauer, 2002
).
Two major regulatory systems involved in the activation of PS gene expression upon a decrease in oxygen availability are (i) the two-component regulatory system, PrrB/PrrA, where PrrA is a response regulator and PrrB is its cognate sensor kinase (Lee & Kaplan, 1992b; Eraso & Kaplan, 1994
, 1995
) and (ii) the anaerobic activator FnrL, a homologue of Escherichia coli Fnr (Zeilstra-Ryalls & Kaplan, 1995
). Both the PrrB/PrrA system and FnrL are global regulators; in addition to activating PS genes, they control expression of a number of other genes in an oxygen-dependent manner (Comolli et al., 2002
; Laratta et al., 2002
; Zeilstra-Ryalls & Kaplan, 1998
).
In addition to the activation of PS gene expression in response to decreased oxygen tension, a repressor PpsR downregulates PS gene expression in the presence of oxygen (Penfold & Pemberton 1991, 1994
). PpsR binds to the target sequences, TGTN12ACA (where N is a nucleotide), located upstream of puc and several crt and bch operons (Gomelsky & Kaplan, 1995a
; Gomelsky et al., 2000
). The DNA binding activity of PpsR is regulated in part through oxidation/reduction of the thiol groups of its cysteine residues (Masuda et al., 2002
; Masuda & Bauer, 2002
) and in part through the anti-repressor AppA (Gomelsky & Kaplan, 1995c
, 1997
, 1998
; Braatsch et al., 2002
; Gomelsky & Klug, 2002
; Masuda & Bauer, 2002
).
The gene encoding AppA anti-repressor was identified by Gomelsky & Kaplan (1995c) in a genetic screen, in which cosmids representing the R. sphaeroides library were identified that upregulated PS gene expression in the PrrB or PrrA mutants when provided in trans. Here we describe the characterization of the second gene identified in this screen. In our preliminary reports this gene was designated ppa for photopigment and puc activation (Gomelsky & Kaplan, 1994
, 1995a
; Horne et al., 1997
; Zelistra-Ryalls et al., 1998
). It will be further referred to as ppaA.
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METHODS |
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Genetic manipulations
Standard recombinant DNA techniques were applied (Sambrook et al., 1989). Plasmid mobilization into R. sphaeroides and P. denitrificans was carried out from E. coli S17-1 as described previously (Gomelsky & Kaplan, 1995a
)
Construction of the PPA1 mutant.
A deletion/substitution mutation in ppaA from R. sphaeroides 2.4.1 was constructed by replacing the internal NcoI fragment with an Kmr cartridge from pUI1637 (Gomelsky & Kaplan, 1995b
) to generate plasmid p714BgH
Nc : : Km (see Fig. 1
b). The mob region and Tcr gene from pSUP202 were cloned into p714BgH
Nc : : Km and the resulting plasmid, p714BgH
Nc : : Km : : mob, was transferred into R. sphaeroides 2.4.1 by conjugation. Several Kmr Tcs exconjugants were selected under anaerobic/dark conditions in the presence of DMSO. The DNA structure of the mutants was confirmed by Southern hybridization. One mutant strain was designated PPA1 and used subsequently.
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Proteinprotein interactions.
Yeast two-hybrid system MatchMaker (Stratagene) was used to assay for PpaAPpsR interactions. Plasmid pYppsR, expressing the full-length PpsR protein translationally fused to the B42 transactivation domain (prey), was constructed based on vector pYESTrp2. A second plasmid, pZppaA, expressing the PpaA protein translationally fused to the DNA-binding domain of the E. coli LexA protein (bait), was constructed based on vector pHybLex/Zeo. As a positive control for proteinprotein interactions, plasmid pZppsR expressing the lexA : : ppsR gene fusion was constructed based on vector pHybLexZeo. The plasmids were introduced into S. cerevisiae L40.
RNA extraction and quantitative RT-PCR.
R. sphaeroides cells in the early exponential phase (OD600 0·160·20) were collected into centrifugation bottles containing shaved ice. Rifampicin was added to a final concentration of 200 µg ml-1 to halt transcription initiation. Cells were pelleted by a brief centrifugation at high speed and cell pellets were frozen at -80 °C until further processing. The cell lysis buffer from the RNAEasy midikit (Qiagen) and an equal volume of sterile zirconium beads were added to the frozen pellets. Cells were disrupted by 1 min of shaking in a Mini-BeadBeater (Biospec Products). RNA was extracted from the supernatants of cell lysates by use of the RNAEasy midikit and tested for the lack of contamination with genomic DNA by quantitative real-time PCR. cDNA from DNA-free RNA was made using SuperScript II reverse transcriptase (Stratagene). The following primers were designed for gene amplification: puc, Puc2AB-F (5'-GGCAAAATCTGGCTCGTGGT-3') and Puc2AB-R (5'-GGTGGTGGTCGTCAGCACAG-3'); puf, PufAB-F (5'-ACATCTGGCGTCCGTGGTTC-3') and PufAB-R (5'-ATCACCGCGAGGAGGAACAG-3'); bchF, BchF2-F (5'-GCGATCTGGGAGGAAGGTGGT-3') and BchF2-R (5'-TGACGCCGAACGAGAAGACA-3'); crtA, CrtA-F (5'-TCACGCTCAGTATCTTCCGGTTC-3') and CrtA-R (5'-CCAGCTTGTTTGCGGTCATCT-3'); rpoZ, OMEGA-F (5'-ATCGCGGAAGAGACCCAGAG-3') and OMEGA-R (5'-GAGCAGCGCCATCTGATCCT-3'). The rpoZ gene (encoding the -subunit of RNA polymerase) was used to normalize expression values for all other genes. In DNA microarray experiments its expression was shown to be independent of growth conditions (data not shown). SYBR Green was used to monitor amplification and to quantify the amount of PCR products using the iCycler iQ Real Time PCR Detection System (Bio-Rad).
DNA microarray experiments.
The construction and methodology of the R. sphaeroides genechips will be described elsewhere (unpublished). The expression data obtained here were deposited in the Gene Expression Omnibus (GEO) database of NCBI (http://www.ncbi.nih.gov/geo) under the following accession numbers: 30 % oxygen, GSM1670 and GSM1671; 3 % oxygen, GSM1672 and GSM1673; 0 % oxygen, GSM2416 and GSM2417.
Quantitative immunoblot.
Frozen pellets of R. sphaeroides cells were resuspended in phosphate-buffered saline (Sambrook et al., 1989) and broken by passage through a French Pressure unit. Soluble proteins (obtained after 30 min centrifugation at 2x104 g) were loaded onto a 0·1 % SDS/10 % polyacrylamide gel, electrophoresed, transferred onto a nitrocellulose membrane and assayed with the PpsR-specific antibody. The anti-PpsR serum was obtained from rabbits injected with the PpsR protein band from SDS-PAGE. The purified PpsR protein was obtained from the overexpressed, affinity purified and site-specific proteolysed GST-PpsR fusion described by Gomelsky et al. (2000)
. Development of immunoblots was performed using a chemiluminescent substrate SuperSignal West Dura (Pierce) according to the protocol of the manufacturer. For quantification of chemiluminescence, the VersaDoc Imaging System with a Nikon camera and Quantity One software (Bio-Rad) were used. To ensure a linear range of chemiluminescence responses, several protein concentrations were loaded on a given gel. Samples of equal protein content from the mutants and the wild-type were loaded in parallel. PpsR protein abundance was calculated based on assigning a chemiluminescence value of the wild-type sample at a given total protein amount to 100 %. The chemiluminescence values of the mutants of the same total protein amount were calculated as percentages of the wild-type values. Mean values were calculated from two parallel measurements (23 different protein concentrations each) from two biological replicates.
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RESULTS |
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To assess the effect of ppaA in extra copy, we extracted photopigments from the PRRA1 strain containing in trans either the ppaA gene on plasmid pSmNo or vector pRK415. At high oxygen tension, when only traces of photopigments are present, the increase in carotenoids and bacteriochlorophyll was observed in strain PRRA1(pSmNo) compared to PRRA(pRK415) (Fig. 2a). At low oxygen tensions, the several-fold increase in photopigments abundance in strain PRRA1(pSmNo) compared to PRRA(pRK415) was also evident (Fig. 2b
).
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PpaA and FnrL.
We tested the effect of ppaA in extra copy on puc : : lacZ expression in the FnrL null mutant, JZ1678(pCF200Km). ppaA in extra copy activated puc expression in this mutant, suggesting that its effect is independent of FnrL (Fig. 3a).
PpaA and bacteriochlorophyll biosynthetic pathway.
Bacteriochlorophyll, or an intermediate in the bacteriochlorophyll biosynthetic pathway, influences expression of PS genes in a related bacterium, Rhodobacter capsulatus (Rödig et al., 1999; Abada et al., 2002
). To investigate whether the effect of PpaA depends on the bacteriochlorophyll biosynthetic pathway in R. sphaeroides, we introduced ppaA in extra copy into the BchE null mutant, BCHE, impaired in one of the early steps of bacteriochlorophyll biosynthesis. PpaA increased puc : : lacZ expression in mutant BCHE (Fig. 3a
). Therefore, neither the presence of bacteriochlorophyll nor an intact bacteriochlorophyll biosynthetic pathway (following the BchE catalysed step) are required for the activity of PpaA.
PpaA and AppA-PpsR system.
ppaA in extra copy activated puc : : lacZ expression in the AppA null mutant, APP11(pCF200Km), suggesting that its effect is independent of the anti-repressor (Fig. 3a).
Does PpaA work through PpsR? Testing for the effect of ppaA in extra copy in the PpsR null mutant, PPS1, would be unfeasible because expression of puc and photopigment biosynthesis genes in this mutant is already derepressed. Furthermore, mutant PPS1 is genetically unstable in the presence of oxygen, apparently because bacteriochlorophyll intermediates produce toxic reactive oxygen species (Gomelsky & Kaplan, 1997). Therefore, we tested for putative PpaA-PpsR relationships using two alternative approaches. (i) We reconstituted the PpsR-mediated repression of puc in a heterologous host. (ii) We tested for direct interactions between PpaA and PpsR using a yeast two-hybrid system.
(i) Does PpaA affect PpsR-mediated repression of puc expression in a heterologous host? Paracoccus denitrificans, a non-photosynthetic relative of R. sphaeroides, is capable of expressing PS genes when these are introduced in trans (Pemberton & Harding, 1987). P. denitrificans lacks specific regulators of PS gene expression, which makes it a convenient host to examine PS gene regulation (Gomelsky & Kaplan, 1995a
, 1997
). We introduced into P. denitrificans a puc : : lacZ fusion (in plasmid pCF400
) and the second plasmid, carrying ppsR in combination with either intact ppaA (plasmid pSmNs) or inactivated ppaA (plasmid pSmNsXc) (Fig. 1b
). Plasmid pSmNsXc differs from pSmNs by a single base pair deletion that results in a frame-shift mutation in the 5' coding region of ppaA. This mutation, designated ppaA(Xc), completely inactivates ppaA as judged by the inability of ppaA(Xc) in extra copy to affect puc : : lacZ expression in R. sphaeroides (data not shown). As anticipated, the level of
-galactosidase in P. denitrificans containing pSmNs was low, 6±1·5 Miller units, indicative of strong repression by PpsR of puc : : lacZ expression. An identical level of expression was observed for plasmid pSmNsXc. Hence, in this experimental design, we could not detect an effect of ppaA on PpsR-mediated repression of puc expression.
We proceeded to test whether PpaA alone can activate puc : : lacZ expression in P. denitrificans. To this end, plasmid pSmNo carrying the ppaA gene was introduced into P. denitrificans containing plasmid pCF400.
-Galactosidase activity in P. denitrificans was equal to 1751±120 Miller units measured as described previously (Gomelsky & Kaplan, 1995a
). A similar value, 1689±30 Miller units, was obtained when vector pRK415 was present in place of pSmNo. Therefore, PpaA alone does not affect puc expression in P. denitrificans.
(ii) Do PpaA and PpsR interact? We tested for a possible PpaA-PpsR interaction by using the yeast two-hybrid system. Strain S. cerevisiae L40 expressing PpsR as bait and PpaA as prey (plasmids pYppsR and pZppaA), remained auxotrophic for histidine, and the -galactosidase level was unchanged [3·2 U (mg protein)-1] when compared to a negative control, strain L40(pYESTrp2, pZppaA). These data argue against direct proteinprotein contacts between PpaA and PpsR.
The data presented above argue against involvement of PpaA in the PpsR-mediated repression. However, they need to be taken with caution because it is possible that a critical cofactor required for PpaA activity was not present in the heterologous host (see Discussion).
Construction of the ppaA mutants
The role of PpaA in regulation of PS gene expression was further investigated by constructing ppaA null mutants. Two mutant strains, PPA1 and PPAXc, were constructed. PPA1 contains an Kmr cassette that replaced an internal ppaA fragment between the two NcoI sites (Fig. 1c
). The PPA1 strain had a phenotype that was very similar to the phenotype of the PpsR mutant, PPS1 (Gomelsky & Kaplan, 1997
), i.e. highly pigmented and genetically unstable in the presence of oxygen (data not shown). Penfold & Pemberton (1994)
suggested that the ppsR gene promoter is located between the NcoI sites used for
Kmr cassette insertion. It is likely that an
Kmr cassette in strain PPA1 has a polar effect on the downstream ppsR gene, thus making PPA1 in effect a double ppaA ppsR mutant. This hypothesis was confirmed by greatly diminished levels of the PpsR protein in the PPA1 mutant (immunoblot data not shown).
To avoid a polar effect on ppsR, a ppaA(Xc) frame-shift mutant allele was introduced into the R. sphaeroides genome replacing the wild-type ppaA gene and yielding strain PPAXc. Because the ppaA(Xc) mutation lies upstream of the putative promoter/operator region of ppsR, we anticipated that it would have no effect on ppsR gene expression (Fig. 1c). To test this prediction, we quantified the PpsR protein levels in mutant PPAXc by immunoblot with the PpsR-specific antibody. The PpsR levels in strain PPAXc were essentially identical to those in the wild-type, i.e. 81±14 % at high oxygen tension, 106±20 % at low oxygen tension (Fig. 4
) and 90±17 % at no oxygen (anaerobic photosynthetic conditions), where 100 % represents a value of the wild-type at a given growth condition.
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(i) The photopigment content in PPAXc and the wild-type strain, 2.4.1, grown at low or no oxygen were very similar. However, at high oxygen tension, when photopigments are present only at the background levels, photopigment levels in the PPAXc strain were lower than in the wild-type (Fig. 5a). This suggests that PpaA is required to maintain the low basal level of photopigments at high oxygen tension and is consistent with the stimulatory effect of ppaA in extra copy on photopigment production (Fig. 2a
).
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Interestingly, the genechip data suggest that expression of the ppsR gene is independent of oxygen tension (Fig. 7a). This correlates well with the ppsR : : lacZ data presented by us previously (Gomelsky & Kaplan, 1998
). Therefore, ppaA and ppsR appear have independent promoters and the promoter upstream of ppaA does not contribute significantly to expression of the downstream ppsR gene.
The PpaA protein family
Corrected sequence of the PpaA homologue from R. sphaeroides strain RS630.
The locus upstream of ppsR had previously been identified by Penfold & Pemberton (1994) in R. sphaeroides strain RS630 and designated ppsS (GenBank accession no. L19596). The N-terminal sequence corresponding to PpsS from strain RS630 differed significantly from that of PpaA from strain 2.4.1. It was surprising that two strains of R. sphaeroides would possess such different proteins. We resequenced DNA upstream of ppsR in strain RS630. The newly obtained DNA sequence from strain RS630 (accession no. L19596) is 99·5 % identical to the sequence of ppaA from 2.4.1 resulting in a 100 % identical PpaA protein. Apparently an error in subcloning was involved when initial sequencing was done.
PpaA homologues.
To gain an insight into the potenial function of the PpaA protein, we analysed sequences of PpaA homologues from several species of anoxygenic phototrophic proteobacteria. All species of this group, whose PS gene cluster sequences are available, contain homologues of the ppaA gene. The PpaA homologues share 2640 % identity to each other (Fig. 8). This is consistent with a conserved role for PpaA in PS formation.
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The conserved sequence pattern, DxHxxG(41)xSxL(26-28)xGG, where x is an amino acid, has been found in a large subset of B12-binding enzymes (reviewed by Ludwig & Matthews, 1997). Three residues, D, H and S, form the so-called ligand triad, with H directly co-ordinating cobalt (Fig. 8
). With the exception of L, most of the residues involved in corrinoid binding (Drennan et al., 1994
; Stubbe, 1994
) are either present in the PpaA homologues and ORF10 and ORF11, or substituted for similar residues (Fig. 8
). The presence of E in place of the conserved D in the putative corrinoid-binding pockets of the PpaA homologues is noteworthy. The studies by Amaratunga et al. (1996)
and Jarrett et al. (1996)
showed that substitution of D for E in the B12 binding pocket of the E. coli methionine synthase MetH does not abolish B12 binding. Therefore, sequence analysis strongly suggests that the PpaA proteins bind a corrinoid cofactor(s). The implications of this finding are discussed below.
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DISCUSSION |
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In trying to understand the function of the PpaA protein we encountered the following paradoxes: (i) ppaA expression is highest under anaerobic conditions, therefore one would anticipate that the ppaA gene product is active under these conditions. However, no distinct phenotype related to PS formation of the ppaA mutant was observed under the anaerobic conditions tested. Only in the presence of oxygen was the phenotype of the ppaA null mutant expressed. (ii) The R. sphaeroides PpaA protein acts as an apparent activator of puc expression and photopigment production under aerobic conditions. In contrast, the PpaA homologue from R. capsulatus, AerR (ORF192), was recently reported to function as an aerobic repressor of a subset of PS genes (Dong et al., 2002). In a search for clues regarding PpaA function, we turned to the protein sequence analysis. Based on the alignment of several PpaA homologues, we found that PpaA possibly contains a corrinoid-binding domain. This means that the activity of PpaA may depend on the availability, structure or redox status of the bound corrinoid cofactor. There is as yet no direct evidence of corrinoid binding by PpaA; however, a substitution of the conserved H-residue, a putative co-ordinating ligand for cobalt (Fig. 8
), inactivated PpaA function (unpublished data).
What are the implications of possible corrinoid binding to PpaA? One possibility is that PpaA is an enzyme, or a subunit of a multisubunit enzymic complex. Some bacteriochlorophyll biosynthetic reactions are corrinoid-dependent (Gough et al., 2000), therefore PpaA may be involved in bacteriochlorophyll biosynthesis. However, the ppaA null mutant shows only minor impairment in bacteriochlorophyll biosynthesis and only under aerobic conditions (Fig. 5
). Furthermore, the ppaA gene in extra copy activated PS gene expression independently of the bacteriochlorophyll pathway in the BCHE mutant (Fig. 3
). These observations make PpaA an unlikely candidate for a bacteriochlorophyll biosynthetic enzyme.
An alternative possibility is that PpaA is a bona fide regulator of gene expression whose activity depends on a corrinoid cofactor. This would suggest that PpaA is involved in co-ordination of PS gene expression with the availability or status of corrinoid. Because vitamin B12 is required for bacteriochlorophyll biosynthesis, a co-ordination of PS gene expression with availability of B12 would seem reasonable. Interestingly, evidence from R. capsulatus suggests that intact B12 biosynthetic pathway is needed for correct regulation of PS gene expression (Pollich et al., 1993; Pollich & Klug, 1995
; Rödig et al., 1999
).
To our knowledge, no precedent of a corrinoid-binding sensory protein has yet been documented, whereas other cofactors, e.g. haems, flavins, ironsulfur clusters, have been shown to perform sensory functions, in addition to catalytic functions (Beinert & Kiley, 1999; Christie & Briggs, 2001
; Gilles-Gonzalez, 2001
; Braatch et al., 2002
; Masuda & Bauer, 2002
; Gomelsky & Klug, 2002
). Two groups hypothesized that corrinoids might regulate activities of transcription factors (Roof & Roth, 1992
; Sheppard & Roth, 1994
; Cervantes & Murillo, 2002
). The latter report is especially relevant because it considers a possibility that DNA-binding activity of M. xanthus ORF10 (CarA) might be corrinoid-dependent. The central region of ORF10 shares significant similarity with the PpaA proteins (Fig. 8
). ORF10 is a transcriptional regulator, whose DNA-binding motif resembles a well characterized, MerR-like, helixturnhelix domain (Cervantes & Murillo, 2002
). In contrast to ORF10, no DNA-binding motif could be identified in PpaA or its homologues. However, the R. capsulatus AerR protein was shown to bind to DNA in vitro when present at high concentrations and to assist in DNA bending. It is worth mentioning that the predicted high pI values of the PpaA homologues might contribute to the relatively non-specific binding to negatively charged DNA. If DNA binding by the PpaA homologues is proven to be physiologically meaningful, it would strengthen the hypothesis that these proteins are corrinoid-dependent transcription regulators. It is possible that the differences in the original observations and interpretations of functions of the R. sphaeroides PpaA and R. capsulatus AerR proteins will turn out to be minor when a deeper understanding of the functions of these proteins is achieved.
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ACKNOWLEDGEMENTS |
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Received 30 August 2002;
revised 4 November 2002;
accepted 6 November 2002.