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
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ABSTRACT |
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The GenBank accession number for the osp gene sequence reported in this article is AF547169.
Present address: Department of Microbiology, Pusan National University, Pusan, Korea.
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INTRODUCTION |
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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 cbb3PrrBA 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 cbb3PrrBA 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.
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METHODS |
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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
. 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 pCCON4 (pLO1 derivative) and R. sphaeroides RDXB
.
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 EcoRINdeI 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 LuriaBertani 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
-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 [
-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 [-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 DNAprotein 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 -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.
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RESULTS |
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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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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 -galactosidase activity were detected in this strain under all conditions tested (Fig. 6
).
-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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DISCUSSION |
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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 AppAPpsR anti-repressorrepressor 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.
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ACKNOWLEDGEMENTS |
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Received 20 September 2002;
revised 9 December 2002;
accepted 16 December 2002.