Digging deeper: uncovering genetic loci which modulate photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

Jeong-II Oh{dagger}, In-Jeong Ko and Samuel Kaplan

Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, Medical School, 6431 Fannin, Houston, TX 77030, USA

Correspondence
Samuel Kaplan
samuel.kaplan{at}uth.tmc.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A new genetic locus was identified in Rhodobacter sphaeroides which is required for optimal synthesis of the light-harvesting spectral complexes as well as for optimal growth under anaerobic conditions with dimethyl sulfoxide (DMSO) as a terminal electron acceptor. The primary structure of the deduced osp gene product shows significant homology to the receiver domain of known response regulators common to bacterial two-component systems. However, site-directed mutagenesis revealed that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway. Paradoxically, the effect of the Osp protein upon spectral complex levels is exerted at the transcriptional level of photosynthesis gene expression. The absence of the Osp protein does not appear to have a general effect on house-keeping metabolism. In cells lacking Osp, the levels of DMSO reductase appear to be normal. The quaternary structure of the Osp protein was determined to be a homodimer and it was directly demonstrated that Osp does not bind to the promoter region of photosynthesis genes as judged by mobility-shift experiments and primary structure analysis.


Abbreviations: PS, photosynthesis

The GenBank accession number for the osp gene sequence reported in this article is AF547169.

{dagger}Present address: Department of Microbiology, Pusan National University, Pusan, Korea.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Rhodobacter sphaeroides is a purple, non-sulfur bacterium that is able to grow photosynthetically under anaerobic conditions in the presence of light. The synthesis of spectral complexes and their associated photopigments (bacteriochlorophyll and carotenoid) occurs under oxygen-limiting conditions (below ~3 % O2) and anaerobiosis. Therefore, photosynthesis (PS) genes encoding the apoproteins of the reaction centre and light-harvesting complexes I (B875) and II (B800-850) (puhA, puf and puc), as well as the genes (bch and crt) involved in photopigment biosynthesis, are strictly regulated by oxygen tensions in the growth environment (Kiley & Kaplan, 1988; Zeilstra-Ryalls et al., 1998; Oh & Kaplan, 2001).

One of the several major regulatory systems involved in the regulation of PS gene expression in response to changes in oxygen tension is the PrrBA two-component activation system (Lee & Kaplan, 1992; Eraso & Kaplan, 1994, 1995, 1996, 2000). This system is required for the activation of many PS genes and also participates in the control of genes involved in CO2, N2 and H2 utilization, as well as of those genes encoding elements of the respiratory electron transport chain (Joshi & Tabita, 1996; Qian & Tabita, 1996; Elsen et al., 2000; Swem et al., 2001). Recently, it was reported that the PrrBA system also positively regulates the che operon 2 of R. sphaeroides (Romagnoli et al., 2002). The PrrBA two-component system consists of the membrane-associated PrrB sensory histidine kinase and its cognate PrrA response regulator. The PrrB histidine kinase is a bifunctional enzyme which has kinase and phosphatase activities (Comolli et al., 2002; Potter et al., 2002). The intrinsic state of PrrB is in the kinase-dominant mode, i.e. in the absence of the inhibitory signal to PrrB, the net activity of PrrB is in favour of the kinase activity rather than the phosphatase activity (Oh et al., 2001; Potter et al., 2002). Inactivation of the cbb3 cytochrome c oxidase in R. sphaeroides leads to derepression of those genes that are regulated by the PrrBA two-component system, even under highly aerobic conditions (Zeilstra-Ryalls & Kaplan, 1996; O'Gara et al., 1998). This observation as well as several additional lines of genetic and biochemical evidence enabled us to suggest that the PrrBA two-component system resides downstream of the cbb3 oxidase in a signal transduction pathway (Oh & Kaplan, 1999, 2000; Oh et al., 2000). In this pathway, the cbb3 oxidase generates a signal under aerobic conditions which shifts the relative equilibrium of PrrB activity from the kinase mode to the phosphatase-dominant mode, resulting in the repression of PS gene expression under aerobic conditions. It was suggested that the extent of electron flow through the cbb3 cytochrome c oxidase determines the relative activity of PrrB: the greater the electron flow through the cbb3 oxidase, the more favoured is the phosphatase-dominant mode of PrrB (Oh & Kaplan, 2000).

Immediately downstream of the ccoNOQP operon encoding the cbb3 oxidase is located the rdxBHIS operon which is required for the synthesis of the active cbb3 oxidase (Roh & Kaplan, 2000). In addition to the cbb3 oxidase, the rdxB gene product appears to be involved in the cbb3–PrrBA signal transduction pathway, since an in-frame deletion mutant of rdxB with intact cbb3 oxidase activity synthesizes spectral complexes under highly aerobic conditions as observed for Cco- strains (Roh & Kaplan, 2000). In order to expose additional regulatory elements which either are related to the cbb3–PrrBA signal transduction pathway or act as an activator in the regulation of PS gene expression, Tn5 random mutagenesis was performed in the background of a CcoN- RdxB- strain in which PS gene expression is fully turned on even under highly aerobic conditions. It was reasoned that in this genetic background other regulatory elements contributing to PS gene expression could be uncovered through subsequent mutagenesis.

In this study, we report the identification of a new genetic locus which is required for the optimal activation of PS genes in R. sphaeroides.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. R. sphaeroides and Escherichia coli strains were grown as described previously (Oh et al., 2000).


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations and conjugation techniques.
Standard protocols (Sambrook et al., 1989) or manufacturer's instructions were followed for general recombinant DNA manipulations. Introduction of plasmids into R. sphaeroides strains was performed by conjugation as described previously (Davis et al., 1988).

Transposon mutagenesis.
The mobilizable suicide plasmid pSUPTn5TpMCS was introduced into R. sphaeroides CcoNRdxB by mating with E. coli S17-1 containing the plasmid. The plasmid carries a Tn5 derivative (Tn5TpMCS) encoding trimethoprim (Tp) resistance (Choudhary et al., 1994). Tn5-insertion mutants of R. sphaeroides CcoNRdxB were selected for their Tp resistance on Sistrom's medium A (SIS) plates incubated aerobically in the dark. Colonies with less pigmentation, when compared to the parental strain CcoNRdxB, were isolated and their ability to grow under photosynthetic conditions at medium light intensity (10 W m-2) was tested. Only those strains that could grow under photosynthetic conditions were selected and further characterized.

Characterization of transposon insertion.
From the Tn5-insertion mutants that grew under photosynthetic conditions total chromosomal DNA was isolated as described previously (Ausubel et al., 1988). Total DNA was completely digested with EcoRI and ligated into EcoRI-digested pUC19. The resulting recombinant plasmid was used to transform E. coli DH5{alpha}. Since pSUPTn5TpMCS has a unique EcoRI site outside the Tp-resistance gene, one of two (left and right) DNA regions flanking the Tn5 insertion was cloned by selecting the E. coli clones for Tp and ampicillin resistance. Using primer Tn5 (5'-TTCAGGACGCTACTTGTGTA-3') the DNA sequence of the region flanking the Tn5 insertion was determined.

Construction of plasmids
pR746-1 and pR746-2.
A 746 bp fragment including the osp gene was amplified with primers CT18+ (5'-CGGCTCCTGCAGGCGGCAGGCGGCCGC-3') and CT18- (5'-ATGAACGGATCCCTGGTGCTCAAAATC-3') using chromosomal DNA isolated from strain 2.4.1 as the template and Takara LA Taq DNA polymerase (Panvera). The PCR product was cloned into pGEM-T Easy vector to yield pGEM : : 746. Following verification of the DNA sequence of the insert by DNA sequencing, a 0·75 kb EcoRI fragment containing the osp gene from pGEM : : 746 was cloned into pRK415 digested with EcoRI, resulting in pR746-1 and pR746-2. Plasmid pR746-1 carries the osp gene in a collinear orientation to lacZ, and pR746-2 has the cloned DNA in a divergent orientation to lacZ.

pCTLACZ.
To construct the osp : : lacZ transcriptional fusion, the promoter region of osp was amplified with primers 5'-TTCAAGCTGCAGGACGCGCGCGGCGC-3' (PstI site is underlined) and 5'-CCACGATCTAGACGTGCATTCTGGCTTC-3' (XbaI site is underlined) and pGEM : : 746 as the template to generate a 307 bp product. The PCR product was digested with PstI and XbaI and cloned into the promoterless lacZ vector pCF1010 digested with the same enzymes, yielding plasmid pCTLACZ.

Construction of a deletion mutation.
For the construction of a deletion mutation of osp, two rounds of PCR were carried out using Pfu Turbo polymerase (Stratagene). With pGEM : : 746 as the template, two primary PCRs were performed with primers CT18XbaI+ (5'-CGGCTTCTAGATGAAGGACGCGACGCGG-3') and CTDEL- (5'-GCAGAGCACGCGCACCGCACGTCTCCACGATCAGGACGTG-3') and with CTDEL+ (5'-CACGTCCTGATCGTGGAGACGTGCGGTGCGCGTGCTCTGC-3') and CT18SphI- (5'-AAAAAGCATGCTATCTGAAAGAATAT-3') to generate two DNA fragments containing a 40 bp overlapping region. The two primary PCR products were used as templates for the secondary PCR, which was performed using primers CT18XbaI+ and CT18SphI-. The 0·6 kb PCR product was restricted with XbaI and SphI and cloned into the suicide vector pLO1. The deletion of 80 bp from osp was confirmed by DNA sequencing. The resulting plasmid pLOOSP1D was transferred from E. coli S17-1 to R. sphaeroides 2.4.1 by conjugation. Heterogenotes of strain 2.4.1, generated by a single recombination event, were selected for their kanamycin resistance, and homogenotes were obtained from the heterogenotes after a second recombination for sucrose resistance as detailed by Oh & Kaplan (1999). The allelic exchange in the homogenotes that produced isogenic osp deletion mutants was verified by PCR with isolated genomic DNA.

For the construction of R. sphaeroides CcoNRdxB, the same protocol as described above was performed using the plasmid pCCON{Delta}4 (pLO1 derivative) and R. sphaeroides RDXB{Delta}.

Site-directed mutagenesis.
Using the QuickChange Site-Directed Mutagenesis kit (Stratagene) with the template plasmid pGEM : : 746, a series of missense mutations were introduced into the plasmid-borne osp gene. Synthetic deoxyoligonucleotides 33-bases long containing an alanine codon (GCC) in place of Asp-51, Asp-55, Cys-93 and Cys-97 in the middle of their sequences were used for mutagenesis. After verification of mutations by DNA sequencing, the 0·75 kb EcoRI fragment containing the mutated osp gene was cloned into pRK415, resulting in pD51A, pD55A, pC93A and pC97A.

Overexpression and purification of the Osp protein.
Using pGEM : : 746 as the template, a 380 bp fragment including osp was amplified by PCR with primers 5'-GACACATATGCACGTCCTGATCGTG-3' (NdeI site is underlined) and 5'-GGTCAGTGGTGGTGATGGTGGTGGGCCGCG-3' (6 histidine codons are underlined). The PCR product was cloned into pUC19 digested with SmaI, resulting in pUC19 : : CT6HIS. After verification of the DNA sequence of osp by DNA sequencing, a 0·39 kb EcoRI–NdeI fragment from pUC19 : : CT6HIS was cloned into pT7-7 digested with the same enzymes, yielding pRCT6HIS.

E. coli Bl21(DE3) carrying pRCT6HIS was grown at 37 °C to an OD590 value of 0·5 in Luria–Bertani medium supplemented with ampicillin. The induction of the osp gene was triggered by addition of IPTG to a final concentration of 1 mM and cells were grown at 30 °C for 4 h. After harvesting of a 1 l culture, cells were resuspended in 25 ml of buffer A (20 mM Tris/HCl, pH 7·9) and disrupted by two passages through a French pressure cell. After the addition of PMSF to a final concentration of 1 mM, the soluble fraction was obtained by centrifugation at 100 000 g for 60 min. After addition of imidazole to the soluble fraction to a final concentration of 5 mM, 2 ml of the 50 % (v/v) nickel-nitrilotriacetic acid HIS-bind slurry (Novagen) were added to the soluble fraction and mixed gently by shaking at 4 °C for 2 h. The protein/resin mixture was loaded into a column and the column was washed with 10 volumes of binding buffer (buffer A with 500 mM NaCl and 5 mM imidazole), 6 volumes of wash buffer I (buffer A with 500 mM NaCl and 60 mM imidazole) and 5 volumes of wash buffer II (buffer A with 500 mM NaCl and 100 mM imidazole). The Osp protein was eluted with the elution buffer (buffer A with 500 mM NaCl and 500 mM imidazole). Fractions containing the Osp protein were dialysed overnight against 2 l of buffer A to remove imidazole and NaCl. The desalted Osp protein was concentrated by means of ultrafiltration using a YM10 membrane (Millipore).

Determination of the native molecular mass of the purified Osp protein.
This was done by Ferguson plot analysis (Ausubel et al., 1988). The purified Osp protein, as well as standard proteins, was subjected to native discontinuous electrophoresis at four different concentrations of acrylamide (10·0, 12·5, 15·0 and 17·5 %). The standard proteins used to construct a molecular mass standard curve (Ferguson plot) were {alpha}-lactalbumin (14·2 kDa), carbonic anhydrase (29 kDa), ovalbumin (45 kDa) and BSA (66 kDa), which were purchased from Sigma.

RNA isolation and analysis.
Total RNA was isolated from R. sphaeroides strains as described by Oelmuller et al. (1990). For Northern hybridization experiments, an appropriate amount of denatured RNA was transferred onto a nylon membrane by vacuum-blotting following electrophoresis on a formamide/agarose gel. Dot-blotting was performed by spotting RNA samples directly onto the nylon membrane by means of a micropipette. DNA probes used in RNA hybridizations were labelled either radioactively with [{alpha}-32P]dCTP (NEN Life Science) using a random primer labelling system (RadPrime DNA Labelling System; Life Technologies) or non-radioactively using the AlkPhos DIRECT system (American Pharmacia Biotech) as instructed by the manufacturer.

Gel mobility-shift assay.
A 0·48 kb EcoRI fragment from pPUF containing the puf promoter region was labelled by filling in recessed 3' ends with [{alpha}-35S]dATP (NEN Life Science) using the Klenow fragment of DNA polymerase I. The enzyme and dNTP were removed by phenol extraction and ethanol precipitation. The DNA–protein binding reaction and non-denaturing PAGE were performed as described previously (Oh & Bowien, 1999).

Quantitative analysis of spectral complexes.
The levels of the B800-850 and B875 complexes were determined spectrophotometrically as described previously (Oh & Kaplan, 1999).

Enzyme assay and protein determination.
Preparation of crude cell extracts and determination of {beta}-galactosidase and DMSO reductase activities were performed as described previously (McEwan et al., 1985; Oh & Kaplan, 1999). Protein concentration was determined by the bicinchoninic acid protein assay (Pierce) using BSA as the standard protein.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the osp gene
Earlier studies revealed that mutations which inactivated either the cbb3 cytochrome c oxidase or the RdxB protein resulted in expression of the PS genes under aerobic conditions as well as showing enhanced expression under routine photosynthetic conditions (Zeilstra-Ryalls & Kaplan, 1996; O'Gara & Kaplan, 1997; O'Gara et al., 1998). We therefore reasoned that in a doubly mutant background (cbb3- RdxB-) it might be possible to identify additional genetic elements modulating PS gene expression by searching for mutant strains showing decreased PS gene expression. We performed Tn5 mutagenesis in a CcoN- RdxB- strain of R. sphaeroides (CcoNRdxB). Strain CcoNRdxB gives rise to dark-red colonies on SIS plates incubated aerobically, since this strain forms spectral complexes even under highly aerobic conditions, unlike the wild-type strain 2.4.1 in which PS gene expression is repressed. Following mobilization of pSUPTn5TpMCS from E. coli S17-1 to R. sphaeroides CcoNRdxB, we isolated colonies formed on SIS plates containing trimethoprim that showed less pigmentation when compared to those of the parental strain CcoNRdxB. We were not interested in mutants unable to grow photosynthetically because we previously found that most mutants incapable of photosynthetic growth had mutations either in those genes encoding the major regulatory systems, such as prrA, fnrL and appA, or in genes involved in bacteriochlorophyll biosynthesis. Three mutants among the 21 selected Tn5-insertion mutants were capable of both photosynthetic growth under medium light conditions (10 W m-2) and anaerobic respiration with DMSO, excluding the possibility that these mutants were impaired in prrBA, fnrL or appA. The sequences of the flanking regions of the Tn5 insertion sites in the mutants were determined as described in Methods, and the resulting sequences were BLAST-searched against the genome sequence of R. sphaeroides 2.4.1. The deduced amino acid sequence from one of the three ORFs containing Tn5 showed significant homology to known response regulator proteins of two-component activation systems. We designated this gene osp (optimal synthesis of the photosynthetic apparatus) and below we have further characterized the gene. The osp gene located on chromosome I encodes a protein consisting of 120 aa. The gene product has a theoretical molecular mass of 13 301 Da and a pI value of 6·13. As shown in Fig. 1, two ORFs neighbour the osp gene in the same transcriptional orientation. One is 243 bp upstream of and the other is 111 bp downstream of the osp gene. The deduced product of orf1 has homology to proteins belonging to the LysR family of regulators (Schell, 1993), whereas that of orf2 does not show any homology to proteins of known function. Downstream of orf2 is located a gene encoding aspartate aminotransferase. A gene encoding a homologue of ribonuclease H1 is located upstream of orf1. The genetic organization of this locus is not conserved nor is there an osp homologue with approximately the same size and E value, of less than 0·01, present in a close relative of R. sphaeroides, Rhodobacter capsulatus, as determined by a BLAST search against the genome sequence of R. capsulatus (at http://wit.integratedgenomics.com/IGwit).



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Fig. 1. Genomic organization of the osp gene and its flanking genes. Tn5 insertion site in osp is indicated by the inverted triangle. The rectangle below the osp gene represents the relative position of the 80 bp deletion introduced into R. sphaeroides OSP1. The gene sizes and lengths of the intergenic regions are given below the diagram (number of nucleotides).

 
Strain CcoNRdxB into which Tn5 was inserted in the osp gene (strain CT18) was cultivated under 30 % O2 conditions, along with the parental strain CcoNRdxB, and its levels of spectral complexes were quantitatively determined. As shown in Fig. 2, the levels of light-harvesting complexes B800-850 and B875 measured for strain CT18 were substantially lower than those found in CcoNRdxB, which is consistent with the pigmentation observed for the colonies of CT18 on SIS plates incubated aerobically. To ensure that CT18 carried no additional mutations other than in osp and that the reduction in spectral complex levels in CT18 was not the result of the polar effect of the Tn5 insertion on the expression of the downstream genes, complementation analyses were performed. Plasmids bearing only the osp gene (pR746-1 and pR746-2) were introduced into strain CT18 and complementation was judged by a return of B800-850 and B875 levels to approximately those found in the parental strain CcoNRdxB under aerobic conditions. Plasmids pR746-1 and pR746-2 are both derivatives of pRK415 and carry DNA inserts containing the osp gene and the intergenic region between orf1 and osp cloned in both orientations. In the case of pR746-1, the osp gene can be expressed from both the tetracycline-resistance-gene promoter on the plasmid and its own promoter if present. Introduction of either pR746-1 or pR746-2 into strain CT18 led to the almost complete complementation of the osp mutation (Fig. 2), indicating that the disruption of the osp gene itself, and not the polar effect of the Tn5 insertion into osp on the gene downstream of osp, is the cause of the reduced levels of spectral complexes in strain CT18 when compared with strain CcoNRdxB. The intergenic region between orf1 and osp may contain the promoter of the osp gene, as judged by the observation that the introduction of pR746-2 into strain CT18 led to complementation of the Osp- phenotype of strain CT18. These initial results provided the impetus for a more detailed analysis of the osp gene and its role in PS gene expression.



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Fig. 2. Levels of spectral complexes in R. sphaeroides strains grown under highly aerobic conditions. Strains were grown aerobically by sparging with 30 % O2, 69 % N2, 1 % CO2 to an OD600 value of between 0·3 and 0·4. The open and shaded bars indicate the levels of B800-850 and B875 light-harvesting complexes, respectively. All values provided are the means of two independent determinations; vertical bars represent the SD from the mean.

 
Phenotypic analyses of an Osp null-mutant
A deletion mutant of osp (strain OSP1) was constructed in the background of the wild-type strain R. sphaeroides 2.4.1. An 80 bp DNA segment was deleted from the 5' portion of the osp gene. The deletion resulted in a frameshift, thereby completely disrupting the synthesis of the osp gene product. As shown in Table 2, under highly aerobic conditions (30 % O2) neither the wild-type nor strain OSP1 synthesized spectral complexes, as opposed to strain CcoNRdxB (see Fig. 2). The formation of spectral complexes, when compared with the wild-type strain 2.4.1 grown under the same conditions, was significantly reduced in strain OSP1 under semi-aerobic (2 % O2) and anaerobic conditions with the exception of high light (100 W m-2) photosynthetic conditions. Under high light photosynthetic conditions, the levels of spectral complexes synthesized in strain OSP1 were comparable to those found for the wild-type strain (Table 2). These results indicated that the osp gene product is required for the optimal formation of the spectral complexes under conditions when high levels of spectral complexes are normally synthesized. To ascertain whether or not the decrease in spectral complex levels was the result of reduced PS gene expression at the transcriptional level, the expression of the PS genes was investigated by means of Northern blotting or dot-blotting analyses using total RNA isolated from the wild-type and strain OSP1. As shown in Fig. 3, puhA, puf and puc, which encode the apoproteins of the reaction centre and the B875 and B800-850 light-harvesting complexes, respectively, were induced in the wild-type and strain OSP1 under semi-aerobic conditions. All transcript levels, especially those for puhA and puc, were significantly decreased in strain OSP1 when compared with the wild-type. Since distinct transcript bands for bacteriochlorophyll genes bchC and bchF were not readily observed in Northern blot experiments, dot-blot analyses were performed using total RNA isolated from the wild-type and strain OSP1 grown semi-aerobically. As with puhA, puf and puc, lower transcript levels corresponding to bchC and bchF were observed in strain OSP1 in comparison with the wild-type. However, transcript levels corresponding to trpA, which encodes the trytophan synthase {alpha}-subunit, appeared to be approximately the same in both the wild-type and strain OSP1 grown under semi-aerobic conditions, suggesting that the absence of Osp does not appear to lead to a generalized transcription defect but instead specifically affects PS gene expression.


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Table 2. Levels of spectral complexes in R. sphaeroides strains grown under various conditions

Strains were grown aerobically (30 % O2) or semi-aerobically (2 % O2), by sparging with the appropriate gas mixture, to an OD600 value of between 0·3 and 0·4. Photosynthetic (PS) cultures were grown at a high (100 W m-2) or low (2·5 W m-2) incident light intensity in completely filled screw-cap glass tubes. Anaerobic growth with DMSO as a terminal electron acceptor was performed in completely filled screw-cap tubes in the dark. The tubes containing anaerobic cultures (PS and dark-DMSO cultures) were rotated by using a rotary drum to keep cells suspended and mixed. Anaerobic cultures were harvested at an OD600 value of between 0·3 and 0·4.

 


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Fig. 3. Comparison of transcript levels of PS genes between the wild-type strain and strain OSP1. Total RNA was isolated from R. sphaeroides strains grown aerobically (30 % O2) or semi-aerobically (2 % O2) to an OD600 value of between 0·4 and 0·5. Approximately 20 µg of total RNA was loaded in each lane in Northern blot analyses. Total RNA (10 µg) was used for dot-blotting. The probes used to detect the puc-, puf- and processed 23S (14S) rRNA-specific transcripts were labelled with a 0·53 kb BamHI–KpnI fragment from pUI624, a 0·47 kb Styl fragment from pUI655 and a 0·5 kb HindIII–PstI fragment from pUCP6.37, respectively. Labelled PCR products, which were generated using internal primers of the corresponding gene, were employed as probes for puhA, bchC, bchF and trpA. The puc- and puf-specific transcripts represent 0·5 kb, small transcripts. Northern blotting with the puhA-specific probe shows two bands with estimated sizes of 1·1 and 1·3 kb.

 
To examine whether the decreased levels of PS gene transcripts in strain OSP1 were the result of differences in the stability of PS gene transcripts when compared to the wild-type, the in vivo stability of the puf-specific mRNA was determined by dot-blot analyses using total RNA isolated from the semi-aerobically grown and rifampicin-treated wild-type strain and strain OSP1. The half-life of the puf transcript for strain OSP1 was identical to that obtained from the wild-type (the half-life of the puf mRNA for both strains was ~30 min, data not shown). Taken together, these results suggest that PS gene expression in strain OSP1 is affected at the transcriptional level, not at the post-transcriptional level.

Growth rates of strain OSP1 and the wild-type strain were not significantly different under aerobic (30 % O2), semi-aerobic (2 % O2) and high light photosynthetic (100 W m-2) growth conditions (Fig. 4a). These results further suggest that the osp gene is both specific for and only modulates PS gene expression. However, the doubling time of strain OSP1 was approximately three times greater under low light (3 W m-2) photosynthetic conditions than that of the wild-type, which can be explained by the significantly decreased levels of spectral complexes in the mutant strain. When grown anaerobically in the dark with DMSO as a terminal electron acceptor, strain OSP1 showed approximately the same growth rate as the wild-type until mid-exponential phase (Fig. 4b). However, as the cell culture density increased, growth of strain OSP1 became severely compromised, reaching the stationary phase with only approximately 50 % of the cell density shown by the wild-type. Since the dor operon encoding the DMSO reductase is regulated by FnrL and the PrrBA two-component system, in a redox-dependent manner like many of the PS genes (Zeilstra-Ryalls et al., 1997; Mouncey & Kaplan, 1998; Eraso & Kaplan, 2000), it is possible that the synthesis of DMSO reductase was affected in strain OSP1. To ascertain this possibility, DMSO reductase activity was determined in both the wild-type strain and strain OSP1 grown to stationary phase under dark-DMSO conditions. The DMSO reductase activities detected in strain OSP1 were shown to be the same as those found in the wild-type (Fig. 4b, inset), indicating that cessation of growth of strain OSP1 at lower cell densities in comparison to the wild-type did not result from any reduction of DMSO reductase activity in the mutant.



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Fig. 4. Growth rate of the wild-type (2.4.1) and strain OSP1 under various growth conditions (a) and the growth curve under dark-DMSO conditions (b). The strains were grown aerobically (30 % O2) or semi-aerobically (2 % O2) by sparging with appropriate gas mixture as described previously (Oh et al., 2000). Photosynthetic cultures were grown at a high (100 W m-2) or low (2·5 W m-2) incident light intensity in completely filled screw-cap glass tubes. Anaerobic growth with DMSO as a terminal electron acceptor was performed in completely filled screw-cap tubes in the dark. The tubes containing anaerobic cultures (photosynthetic and dark-DMSO cultures) were rotated by using a rotary drum to keep cells suspended and mixed. The Klett unit can be converted to the optical density at 660 nm using the following equation: A600=(Klett unit+3·06)/102·04. DMSO reductase activity was measured from cells harvested in the stationary phase.

 
Features of the primary structure of Osp
Most response regulators of bacterial two-component systems consist of two domains, a conserved N-terminal regulatory (receiver) domain and a variable C-terminal effector (output) domain (West & Stock, 2001). The primary structure of Osp exhibits sequence similarities to the CheY-like proteins and other regulatory domains of response regulators (44·2 % similarity to CheY of E. coli) (Fig. 5a). The Osp protein consists of 120 aa and lacks a C-terminal effector domain, which places it into the CheY family of response regulators. The aspartate residue (Asp-51 of Osp) corresponding to the phosphorylation sites of other response regulators (Sanders et al., 1989; Comolli et al., 2002) is conserved in Osp. In addition, two aspartate residues (Asp-12 and Asp-13 in E. coli CheY) that are conserved amongst known response regulators and are known to be involved in co-ordination of the Mg2+ ion that is essential for phosphorylation and dephosphorylation (Lukat et al., 1990; Stock et al., 1993) are not present in Osp. The amino acid corresponding to Asp-12 of E. coli CheY is conservatively substituted by Glu-7 in Osp, whereas Asp-13 of E. coli CheY, which directly coordinates Mg2+, is not conserved in Osp. Thr-87, Tyr-106 and Lys-109 of the E. coli CheY protein were demonstrated to be involved in propagation of a conformational change upon phosphorylation (Lukat et al., 1991; Cho et al., 2000). The threonine and tyrosine are conserved in Osp, while lysine is substituted by isoleucine in Osp. To ascertain whether Osp is part of a phospho-relay signal transduction system and the conserved Asp-51 is necessary for its function, we performed site-directed mutagenesis experiments. The functionality of the mutant forms of Osp was probed by means of complementation. As shown in Fig. 5(b), when plasmid pR746-2, containing the wild-type osp gene, was introduced into R. sphaeroides CT18 as a positive control, the levels of spectral complexes in strain CT18 grown under dark-DMSO conditions were restored to those measured in the parental strain of CT18, CcoNRdxB, which was indicative of full complementation of the Osp- phenotype. Replacement of Asp-51 with alanine did not affect the functionality of Osp as judged by the observation that the introduction of plasmid pD51A, which is the same construct as pR746-2 with the exception of the D51A replacement in Osp, into strain CT18 led to full complementation. Since there is another aspartate residue (Asp-55) in the vicinity of Asp-51 in Osp, it is possible that Asp-55 of Osp is the alternative phosphorylation site. In order to examine this possibility, complementation tests were performed using plasmids pD55A and pD5155A, in which Asp-55 of Osp and both Asp-51 and Asp-55 are substituted by Ala, respectively. Strain CT18 containing either pD55A or pD5155A showed approximately the same levels of spectral complexes as detected for the positive control strain CT18(pR746-2), implying that neither Asp-51 nor Asp-55 are required for Osp function and that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway.



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Fig. 5. Multiple alignment of the Osp protein and its homologues (a) and complementation tests to determine the functionality of a series of mutant forms of Osp (b). (a) The identical and conservatively substituted residues are indicated by asterisks and colons or periods, respectively. The ‘+’ marks indicate functionally important residues which are well conserved in all known response regulators of the bacterial two-component system. The residues of Osp that were subjected to site-directed mutagenesis are shaded grey. E.c., E. coli; R.s., R. sphaeroides. (b) For complementation tests, strains were grown anaerobically with DMSO as an external electron acceptor in the dark. The values provided are the means of two independent determinations ±SD.

 
The Osp protein contains the sequence motif H92–C93–X–X–X–C97 at its C terminus and its inverted sequence is identical to the known haem c binding motif found in many cytochrome c-like proteins (C–X2–3–C–H) (Rios-Velazquez et al., 2001). To examine whether this motif is important for Osp function, Cys-93 and Cys-97 were individually mutagenized to Ala and the functionality of the mutant forms of Osp was probed by complementation. When either Cys-93 or Cys-97 was replaced with Ala, the mutant forms of Osp were completely functional as evidenced by complementation of the Osp- phenotype by introduction of plasmids pC93A and pC97A into strain CT18 under dark-DMSO conditions, again indicating that neither Cys-93 nor Cys-97 are required for Osp function. Taken together, our sequence analysis and site-directed mutagenesis data suggest that the Osp protein is not a component of a histidine–aspartate phospho-relay signal transduction system nor does it possess functionally important cysteine residues in the 93–97 region, despite the fact that its overall amino acid sequence shows significant homologies to the regulatory domains of response regulators of the bacterial two-component system.

Expression of osp
The transcription rate of the osp gene under several growth conditions was determined by means of an osp : : lacZ transcriptional fusion. The transcriptional fusion plasmid pCTLACZ was constructed from the promoterless lacZ vector pCF1010 and contains the 5' portion of osp and a 281 bp upstream region of osp encompassing the 243 bp intergenic region between osp and orf1. The wild-type strain, R. sphaeroides 2.4.1, carrying pCTLACZ was grown aerobically (30 % O2), photosynthetically at medium light intensity (10 W m-2) or anaerobically under dark-DMSO conditions. R. sphaeroides 2.4.1(pCF1010) served as a negative reference, and only basal levels of {beta}-galactosidase activity were detected in this strain under all conditions tested (Fig. 6). {beta}-Galactosidase activities measured for R. sphaeroides 2.4.1(pCTLACZ) were low but well above those detected in the negative control strain. Expression of osp was constitutive under the tested growth conditions, although the promoter activity of osp was slightly lower under highly aerobic conditions than that measured under anaerobic growth conditions (photosynthetic and dark-DMSO conditions). From these results, we conclude that osp is expressed constitutively at a low level under both aerobic and anaerobic conditions.



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Fig. 6. Promoter activities of the osp gene in R. sphaeroides 2.4.1 grown under various growth conditions. (a) Schematic diagram of osp : : lacZ transcriptional fusion plasmid pCTLACZ. (b) Wild-type (2.4.1) strain containing either pCF1010 (shaded bars) or pCTLACZ (open bars) was grown aerobically (30 % O2), photosynthetically at medium light intensity (PS, 10 W m-2) or anaerobically with DMSO in the dark (Dark DMSO). Aerobic and anaerobic (PS and Dark DMSO) cultures were harvested at OD600 values of 0·3 and 0·4, respectively. All values provided are the means of two independent determinations; vertical bars represent the SD from the mean.

 
Purification of Osp from E. coli
The osp gene containing six histidine codons immediately before its stop codon was overexpressed in E. coli by means of the T7-promoter system on the expression vector pT7-7. The histidine-tagged Osp protein purified through affinity chromatography showed an apparent molecular mass of ~12 kDa, which correlates relatively well with the calculated molecular mass of the osp gene product including six histidines (14·2 kDa) (Fig. 7a). Western blotting showed that the purified protein cross-reacted with anti-His4 antibody, indicating that the purified protein is the osp gene product (data not shown). The addition of six histidine residues at the C terminus of Osp was not detrimental to Osp functionality, since the introduction of the osp gene with six histidine codons into strain CT18 [CT18(pRCT6HIS)] led to complete complementation of the Osp- phenotype of strain CT18 when spectral complex formation was measured under dark-DMSO growth conditions (Fig. 5b). The molecular mass of native Osp with the histidine tag was found to be 29 kDa, as determined by Ferguson plot analysis (Fig. 7b). Considering the calculated molecular mass of the monomeric Osp protein with six histidine residues, native Osp appears to have the quaternary structure of a homodimer. It has been reported that the regulatory domains of some response regulators, including NtrC and PhoB, serve as dimerization domains and that the effector domains prevent the dimerization of their cognate regulatory domains when the regulatory domains are not phosphorylated (Fiedler & Weiss, 1995). Either phosphorylation of the receiver domain or removal of the effector domain was shown to lead to the dimerization of NtrC and PhoB. Since the primary structure of Osp is made up of only the domain structure similar to the regulatory domain of the response regulator, the quaternary structure of Osp corroborates the suggestion that the regulatory domain of the response regulator is a potential dimerization domain.



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Fig. 7. Purification of the Osp protein and determination of its molecular mass. (a) SDS-PAGE of the purified Osp protein. Proteins were stained with Coomassie brilliant blue R-250. The Osp protein is indicated by the arrow. (b) Ferguson plot. Kr is the slope of the line generated by linear regression when log10 relative mobility of each protein is plotted against gel concentration. CA, carbonic anhydrase.

 
To examine whether Osp was capable of binding to the promoter regions of those PS genes whose expression was shown to be negatively affected in the absence of Osp, gel retardation experiments were performed using the purified Osp protein as well as the 482 bp DNA fragment containing the promoter region of the puf operon. When 0·23 pmol of a 35S-labelled DNA fragment of the puf control region and up to 310 pmol of Osp were employed in 10 µl of binding reaction, no band shift was observed in non-denaturing PAGE using a Tris/acetate buffer system (data not shown). When the same experiment was performed with purified PrrA, a response regulator known to bind to the control region of the puf operon, the addition of 137 pmol of unphosphorylated PrrA to the binding reaction led to a complete shift of 0·23 pmol of the puf control region in a PAGE gel (data not shown). These results imply that although the Osp protein is required for the optimal expression of many PS genes at the transcriptional level, it appears not to be a DNA-binding, trans-acting element, which is consistent with the fact that Osp contains only the counterpart of the regulatory domain of the response regulator, and not the effector domain.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The observation that spectral complex formation in strain CT18 grown under highly aerobic conditions is negatively affected, in comparison with its parental strain CcoNRdxB grown under the same conditions, suggests that Osp is required for the optimal syntheses of spectral complexes not only under oxygen-limiting conditions but also under highly aerobic conditions (30 % O2). This also suggests that the reduced levels of spectral complexes in strain OSP1 grown under semi-aerobic or anaerobic conditions are not due to the negative effect of the lack of Osp upon general house-keeping metabolism under anaerobic conditions. This suggestion is further supported by the following observations: (i) the growth rates of the wild-type strain and strain OSP1 are approximately the same under aerobic, semi-aerobic and high light photosynthetic conditions; (ii) under semi-aerobic growth conditions, the expression of the PS genes, such as puhA, puc, puf, bchC and bchF, is significantly reduced in strain OSP1 when compared with the wild-type, while trpA is expressed at a similar level in both the wild-type strain and strain OSP1.

With regard to the underlying mechanism by which the Osp protein affects PS gene expression, several possibilities are plausible. (i) Osp might be required for optimal operation of the regulatory systems known to be involved in PS gene regulation, such as the PrrBA two-component system, the AppA–PpsR anti-repressor–repressor system (Gomelsky & Kaplan, 1997) and FnrL (Zeilstra-Ryalls & Kaplan, 1995). (ii) The expression of many PS genes, including the genes encoding the apoproteins of the reaction centre and light-harvesting complexes, is negatively affected in mutant strains defective in bacteriochlorophyll biosynthesis, which is indicative of the relationship between the cellular levels of bacteriochlorophyll and PS gene expression (Rödig et al., 1999; Abada et al., 2002). From this observation, it is conceivable that Osp might be implicated in bacteriochlorophyll biosynthesis at either transcriptional or post-transcriptional levels. (iii) When grown under anaerobic conditions with DMSO as an external electron acceptor in the dark, strain OSP1 shows the same growth rate in the early growth phase as does the wild-type. However, the growth of strain OSP1 becomes increasingly retarded from mid-exponential phase and eventually stops at a lower cell density compared to the wild-type grown under the same conditions, despite similar levels of DMSO reductase activity. However, when strain OSP1 was grown anaerobically with DMSO in the presence of high light, no defect in growth was observed (data not shown). PS enables strain OSP1 to overcome the problem(s) imposed by anaerobic respiration with DMSO at the late growth phase. We know that the higher the light intensity under anaerobic photosynthetic conditions the more oxidized is the cellular redox state of R. sphaeroides (Parson, 1975; Oh & Kaplan, 2001). This fact leads us to suggest that Osp might be required for growth of R. sphaeroides when its cellular redox state is poised at very reduced conditions. As DMSO is reduced to dimethyl sulfide (DMS) by DMSO reductase in batch culture of R. sphaeroides, the level of DMS is increased and the level of DMSO is decreased, leading to a decrease in the turnover rate of DMSO reductase. This makes the cellular redox state very reduced, since DMSO reductase is the only terminal reductase under this growth condition that can siphon electrons from the respiratory electron transport chain (McEwan, 1994). Strain OSP1 might be more sensitive to these reduced conditions than the wild-type strain. This hypothesis could explain the growth phenotype that strain OSP1 shows under dark-DMSO conditions. In agreement with this rationale, when ethanol, a far more reduced substrate than succinate, was supplied as the sole electron donor for photosynthetic growth at high light intensity, strain OSP1 grew significantly more slowly than the wild-type (doubling time: wild-type=26·7 h; strain OSP1=40·2 h).

In conclusion, we identified a gene, osp, in R. sphaeroides which is required for optimal synthesis of spectral complexes as well as for optimal growth under dark-DMSO conditions. The effect of the Osp protein upon the levels of spectral complexes is exerted at the transcriptional level of PS gene expression, and the absence of Osp does not appear to have a general effect on house-keeping metabolism. The Osp protein does not appear to be a trans-acting element which can bind to promoter (control) regions of PS genes, as judged by mobility-shift experiments and primary structure analysis. However, we cannot rule out the possibility that Osp acts together with other regulatory proteins. The mechanism by which Osp affects PS gene expression remains to be solved. This study and an earlier study by Sabaty & Kaplan (1996) indicate that although the broad outlines of PS gene regulation are becoming well understood, numerous other effectors clearly exist and remain to be investigated.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grant GM15590 and DOE grant ER63232-1018220-0007203.


   REFERENCES
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DISCUSSION
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Received 20 September 2002; revised 9 December 2002; accepted 16 December 2002.