Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Hiroyuki Arai
aharai{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Hydrogenovibrio marinus strain MH-110 is an obligately lithoautotrophic, halophilic and aerobic hydrogen-oxidizing bacterium isolated from a marine environment (Nishihara et al., 1989, 1991
). This bacterium fixes CO2 via the CBB cycle. We have shown that H. marinus possesses three types of RubisCO: two form I RubisCOs (CbbLS-1 and CbbLS-2), and a form II RubisCO (CbbM) (Fig. 1
) (Chung et al., 1993
; Yaguchi et al., 1994
; Hayashi et al., 1998
; Yoshizawa et al., 2004
). The ambient CO2 concentration affects the expression of these three RubisCO genes (Yoshizawa et al., 2004
). It is already known that cbbM is expressed constitutively, but the expression levels of cbbM increase at CO2 concentrations above 2 %. cbbLS-1 is expressed at 2 % and 0·03 % CO2 concentrations, but not at 15 % and 0·15 % concentrations. cbbLS-2 is expressed at CO2 concentrations below 0·15 %. We have previously shown that the expression of these RubisCO genes is regulated at the transcriptional level (Yoshizawa et al., 2004
), although the molecular mechanism for the regulation has not yet been elucidated.
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The LTTR is known to recognize a specific effector molecule, which alters the ability of the LTTR to bind to the promoter region of its target gene (Schell, 1993). It has been reported that NADPH enhances the DNA-binding ability of the CbbR of Xanthobacter flavus (van Keulen et al., 1998
, 2003
). NADPH also changes the binding ability of the CbbR of Hydrogenophilus thermoluteolus (Terazono et al., 2001
). In the case of Ralstonia eutropha, phosphoenolpyruvate (PEP) is found to increase the DNA-binding ability of the CbbR and has a negative effect on RubisCO transcription in vitro (Grzeszik et al., 2000
). It has recently been reported that some metabolites formed by the CBB cycle and some intermediates, including PEP, affect the ability of two CbbRs to bind to target promoter regions in Rhodobacter capsulatus (Dubbs et al., 2004
). The effects of these effectors have not necessarily been observed in the CbbRs of other bacteria (Grzeszik et al., 2000
; Terazono et al., 2001
; Tichi & Tabita, 2002
), which suggests that alternative effectors are recognized by the CbbRs of other bacteria. The concentration of CO2, which is the substrate for RubisCO, affects the expression of RubisCO in many types of bacteria (Sarles & Tabita, 1983
; Jouanneau & Tabita, 1986
; Hallenbeck et al., 1990a
, b
), although the effects of CO2 on the DNA-binding ability of CbbR have not yet been reported. The expression patterns of the three RubisCOs in H. marinus are known to change according to the CO2 concentration (Yoshizawa et al., 2004
), thus indicating that there is a regulatory mechanism that responds to the CO2 concentration. Two CbbRs appear to be involved in such regulation, and these CbbRs could sense the CO2 concentration or particular metabolites produced in cells exposed to various CO2 concentrations. In this study, we report that each CbbR regulates the expression of the adjacent RubisCO gene, and that the CO2 concentration influences the expression of one of the two cbbR genes. We also show that an interactive regulation between the three RubisCOs is operative in H. marinus.
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METHODS |
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CbbRm was purified by fusion with His-tag. To construct an expression plasmid for His6-CbbRm, the cbbRm gene was cloned into pET21c (Novagen). The construct was used to transform E. coli BL21(DE3) (Novagen). The transformant was cultured in LB supplemented with ampicillin at 37 °C. When the OD600 reached 0·7, IPTG was added at a final concentration of 0·5 mM and the cells were cultured for a further 3 h. Cells were harvested by centrifugation and were suspended in a suspension buffer (20 mM sodium phosphate, pH 7·5, 1 M NaCl). The resuspended cells were broken by passing through a French pressure cell (Aminco, Lake Forest, CA) at 110 MPa. Cell extracts were obtained by centrifugation at 4 °C for 25 min at 20 000 g. The cell extracts were loaded onto a 1 ml nickel Hitrap Chelating affinity column (Amersham Pharmacia). The column was washed with 20-bed volume of suspension buffer containing 50 mM imidazole. The His-tagged CbbRm was eluted with suspension buffer containing 200 mM imidazole. The eluted proteins were dialysed twice at 4 °C in a buffer containing 50 mM HEPES (pH 7·8), 200 mM KCl, 10 mM MgCl2, 1 mM DTT, 0·05 mM PMSF and 50 % (v/v) glycerol, and the proteins were stored at 20 °C.
Preparation of cell extracts of H. marinus.
Cells were harvested from the cultures at the late-exponential phase by centrifugation and were suspended in BEMD buffer (50 mM Bicine, 0·1 mM EDTA, 10 mM MgCl2.6H2O, 1 mM DTT, pH 7·8). The cells were disrupted twice using a French pressure cell (Aminco) at 110 MPa and then centrifuged for 25 min at 20 000 g at 4 °C to remove the cell debris. The protein concentrations were determined by the Bio-Rad protein assay method with BSA as standard.
Western immunoblot analyses.
Ten micrograms of cell extract were resolved by SDS-PAGE and transferred to PVDF membranes (Sequi-Blot PVDF membrane, Bio-Rad) by using a semi-dry blotting system (ATTO, Tokyo, Japan). Polyclonal antibodies raised against synthetic oligopeptides encoded by cbbS-1 or cbbS-2 of H. marinus, and against form II RubisCO of H. marinus at a 1 : 1000 dilution were used to detect the three kinds of RubisCO of H. marinus. The blots were developed as described previously (Yoshizawa et al., 2004).
Gel shift assays.
A DNA fragment containing the promoter region of cbbLS-1, cbbLS-2, cbbM, or orf90cbbRm was amplified by PCR using the genomic DNA of H. marinus as a template with the following primers: 5'-TACGAATTCGTTGTGCC-3' and 5'-TCTTTTGGCTCATACTCTGGCATCC-3' for cbbLS-1; 5'-CCGCAAAAGAATTCACC-3' and 5'-CGTTAACCACAGAAGCTTCTTC-3' for cbbLS-2; 5'-CGAATTGGGATCCTAACTTACCC-3' and 5'-AGTGAATTCAGCGTAACG-3' for cbbM; and 5'-AGAAATTGCATGCTGTACG-3' and 5'-GTGGCATAGATTACATGCATTG-3' for orf90cbbRm. These amplified fragments were inserted into pUC119 at the appropriate restriction sites and sequenced to confirm that no mutation was introduced during the PCR. The DNA fragments containing each promoter region were excised from the constructed plasmids by digestion with the appropriate restriction enzymes. The length of each fragment was as follows: cbbLS-1, 357 bp; cbbLS-2, 293 bp; cbbM, 146 bp; and orf90cbbRm, 227 bp. The fragments were labelled by either filling the recessed end with [-32P]dATP (Amersham Pharmacia) using Klenow enzyme or phosphorylating the end of the fragments with [
-32P]ATP (Amersham Pharmacia) using T4 polynucleotide kinase. The labelling reaction using Klenow enzyme was performed in a 50 µl mixture containing 50 ng of the DNA fragment, 100 µM dCTP, 100 µM dGTP, 100 µM dTTP, 20 µCi [
-32P]dATP, 4 U Klenow enzyme, 10 mM Tris/HCl (pH 7·5), 7 mM MgCl2 and 0·1 mM DTT. After the reaction mixture had been incubated at 37 °C for 1 h, 10 µM dNTPs were added to the mixture, which was then incubated for another 5 min. The labelling reaction using T4 polynucleotide kinase was done in a 50 µl mixture containing 100 ng of the DNA fragment, 20 µCi [
-32P]ATP, 10 U T4 polynucleotide kinase, 50 mM Tris/HCl (pH 8·0), 10 mM MgCl2 and 5 mM DTT. The mixture was incubated at 37 °C for 1 h and then heated at 90 °C for 10 min to inactivate kinase. The labelled DNA was purified by using SUPREC-02 (TaKaRa), and stored at 20 °C. Purified CbbR1 or CbbRm was incubated with the labelled DNA fragments (5000 c.p.m.) in a binding buffer containing 10 mM Tris/HCl (pH 8·0), 50 mM NaCl, 0·5 mM DTT, 10 % (v/v) glycerol, 50 µg ml1 poly(dI-dC).poly(dI-dC) (Amersham Pharmacia) and 50 µg ml1 BSA at room temperature for 30 min. The reaction mixture was applied to a 4 % non-denaturing acrylamide gel in Tris/glycine buffer and run at 10 V cm1. The gel was subsequently dried and visualized using a Bio Imaging Analyser (Fujifilm, Tokyo, Japan) or autoradiographed with intensifying screens at 80 °C.
DNA manipulations.
Routine DNA manipulations, including chromosomal DNA isolation, plasmid preparation, restriction endonuclease digestion, agarose gel electrophoresis, fragment ligation and bacterial transformation were performed according to standard methods (Sambrook et al., 1989). Southern blotting procedures were carried out with a Hybond-N membrane (Amersham Pharmacia). The hybridized DNA was detected by a staining reaction, as described previously (Yoshizawa et al., 2004
).
Construction of H. marinus cbbR mutant strains.
To construct the cbbR1 and cbbRm mutants, a coding region of the cbbR1 or cbbRm gene was amplified by PCR using the total DNA of H. marinus as template with the following primers: 5'-GCTGGTACCTACATTGGTTTTGCC-3' and 5'-TAAGGGAATTCTAATAACAAAATCACC-3' for cbbR1; 5'-CAAAAATCTAGACGATGTCG-3' and 5'-GATTATTAGTATAGACGTCGAC-3' for cbbRm. The amplified fragments containing the cbbR1 and cbbRm genes were cloned into pUC119, resulting in pUCR1 and pUCRm, respectively. A 1·2 kb PvuIIStuI fragment from pHSG298, encoding a Km resistance gene, was inserted into the unique BalI and NruI sites within cbbR1 and cbbRm, respectively. The DNA fragment containing cbbR1 : : Kmr or cbbRm : : Kmr of the resulting plasmids was excised by digestion with EcoRI/BamHI or SphI/XbaI, and was cloned into a suicide vector pJP5608 (Penfold & Pemberton, 1992), resulting in pJPKR1 or pJPKRm. These plasmids were transferred to H. marinus strain MH-110 from E. coli strain S17-1
pir (Simon et al., 1983
) by transconjugation as follows. One millilitre of overnight culture of E. coli donors and 1·5 ml of overnight culture of H. marinus recipients were harvested and washed with sterilized water and 0·5 M NaCl, respectively. The cells of H. marinus and E. coli were suspended with 100 µl 0·5 M NaCl and spotted onto membrane filters (0·45 µm pore size, Milipore) on a plate of inorganic medium (Yoshizawa et al., 2004
). The plates were incubated at 37 °C for 1 day in a desiccator (Tokyo Glass Kikai, Tokyo, Japan) after the gas in the desiccator was replaced with a gas mixture (H2 : O2 : CO2=75 : 10 : 15). Transconjugants on the membrane were suspended in 1 ml 0·5 M NaCl and spread on the inorganic plate containing Km. The Kmr colonies were screened for sensitivity to the plasmid-encoded Tc resistance in order to select strains in which the cbbR genes were disrupted by double homologous recombination. One and two Kmr Tcs strains were obtained from cbbR1 and cbbRm transconjugants, respectively. The disruption of each cbbR gene was confirmed by Southern hybridization. The obtained cbbR1 and cbbRm mutant strains, designated dR1 and dRm, respectively, were used for the subsequent experiments. To construct a cbbR1 cbbRm double mutant strain, the cbbR1 gene of strain dRm was disrupted. A 1·8 kb SmaI fragment encoding the Gm resistance gene from pHP45
aac (Blondelet-Rouault et al., 1997
) was inserted into the unique BalI site in the cbbR1 gene on pUCR1. The EcoRIXbaI fragment containing the disrupted cbbR1 gene on the constructed plasmid was excised and cloned into pJP5608, resulting in pJPGR1. E. coli strain S17-1
pir was used to transfer pJPGR1 into H. marinus strain dRm. Four hundred Gmr colonies were screened for Tc sensitivity and two Gmr Tcs clones were obtained. Double homologous recombination was confirmed by Southern hybridization. One of the obtained Gmr Tcs strains was designated ddR and was used in subsequent experiments.
Complementation of cbbR mutants.
Broad-host-range plasmids, pCR1 and pCRm, were constructed by cloning of the cbbR1 fragment from pUCR1 and the cbbRm fragment from pUCRm, respectively, to pHRP309 (Parales & Harwood, 1993). These plasmids were transferred from E. coli JM109 to the cbbR mutants by the triparental mating method with E. coli HB101 containing the mobilizable helper plasmid pRK2013 (Figurski & Helinski, 1979
). The conditions of conjugation were the same as those described above.
RNase protection assays.
The total RNA was isolated from bacterial cells by using ISOGEN (Nippon Gene, Tokyo, Japan), as described previously (Yoshizawa et al., 2004). 32P-labelled RNA probes for the RNase protection assays were generated by in vitro transcription using a MAXIscript kit (Ambion). The DNA fragment containing either the last 107 codons of cbbR1 or the last 110 codons of cbbRm was amplified by PCR from the genomic DNA as a template with the following primers: 5'-GGAAAAGAATTCTGGTATTCG-3' and 5'-CGACTTCTAGAACAGCACTG-3' for cbbR1; and 5'-AGAGAATTCGGATCCGGTATCC-3' and 5'-CCAATCTAGAATTCGGTAAATTTCG-3' for cbbRm. The amplified fragments were cloned into pGEM-3Zf(+), resulting in pRPAR1 for cbbR1 and pRPARm for cbbRm. The cbbR1 probe, a 411 bp fragment containing the last 107 codons of cbbR1, was generated by transcription with the SP6 promoter using EcoRI-digested pRPAR1 as template. The cbbRm probe, a 440 bp fragment containing the last 110 codons of cbbRm, was generated by transcription with the SP6 promoter using BamHI-digested pRPARm as template. Transcription reactions contained 500 µM ATP, GTP and CTP, and 5 µl [
-32P]UTP [800 Ci mmol1 (29600 GBq mmol1), 10 mCi ml1 (370 MBq mmol1); Amersham Pharmacia]. The reactions were carried out for 15 min with SP6 RNA polymerase. After the transcription reaction, full-length transcripts were recovered after separation on an 8 M urea/5 % polyacrylamide gel. RNase protection assays were carried out using the RPAIII kit (Ambion); 10 µg total RNA was used for the reactions with the cbbR1 riboprobes. Together with the cbbRm riboprobes, 20 µg total RNA was used for the reactions. Unhybridized probes were digested with a 1 : 100 dilution of RNase A/RNase T1 mix. The radiolabelled probes were separated on an 8 M urea/5 % polyacrylamide gel and were visualized using a Bio Imaging Analyser (Fujifilm) and by autoradiography.
Primer extension.
Primer extension mapping of the transcription start sites of cbbLS-1, cbbLS-2, cbbM, cbbR1 and cbbRm was performed by using Primer Extension System-AMV Reverse Transcriptase (Promega). The oligonucleotide primers used in the experiments were as follows: 5'-GTCTTTGCCATTTTTCACCTCTTTG-3' for cbbLS-1, 5'-CTAGAGGAGTGTAGTCTGGTGTCC-3' for cbbLS-2, 5'-TCATGTTTGGGCGGTGATCACAAAG-3' for cbbM and 5'-GCGGGCGACAGATTCAAAAATACG-3' for cbbR1. To map the transcription start site of cbbRm, the oligonucleotides cbbRm-1 (5'-TGTTGGCGATACACTCAAAAACC-3') and cbbRm-2 (5'-GATAATTATCGTTGATTTCGTCG-3') were used. The primers were end-labelled with [-32P]ATP (Amersham Pharmacia). One microlitre of labelled primer and 5 µl AMV primer extension 2x buffer (100 mM Tris/HCl, pH 8·3, 100 mM KCl, 20 mM MgCl2, 1 mM spermidine, 2 mM GTP, CTP, ATP and TTP) were added to 5 µl (20 µg) total RNA. The extension reaction mixtures were incubated at 58 °C for 20 min to anneal the primer to the RNA before cooling the reactions to room temperature for 10 min in a TaKaRa PCR thermal cycler MP. A mixture of 2 mM sodium pyrophosphate, AMV primer extension 1x buffer and AMV reverse transcriptase (0·05 U µl1) was added to the annealed reactions and incubated at 42 °C for 30 min. The primer extension products thus obtained were analysed in a 5 % sequencing gel in parallel with DNA sequencing reactions carried out using the same primers as those used in the primer extension experiments. The plasmids carrying the promoter regions were used as templates for the sequencing reactions.
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RESULTS |
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Induction of cbbLS-2 expression at higher CO2 concentrations by disruption of the two cbbR genes
The cbbR1 gene in strain dRm was disrupted in order to construct the cbbR1 cbbRm double mutant strain ddR. Although strain ddR grew at the same rate as MH-110 at CO2 concentrations below 0·15 %, the strain showed impaired growth at CO2 concentrations above 2 % (Fig. 5). The relative growth rate of ddR was decreased to 57 and 72 % of the wild-type strain at 15 and 2 % CO2 concentrations, respectively. The expression of each RubisCO in strain ddR under all CO2 conditions was analysed by Western blotting. As expected, the levels of CbbLS-1 and CbbM were identical to those in strains dR1 and dRm, respectively, i.e. no CbbLS-1 expression and decreased CbbM expression (Fig. 4d
). It was of note that CbbLS-2 was synthesized in strain ddR even at CO2 concentrations above 2 % at which CbbLS-2 was not expressed in the wild-type. The amount of CbbLS-2 in strain ddR at higher CO2 concentrations was comparable to that at lower CO2 concentrations. The impaired growth of strain ddR at higher (
2 %) CO2 concentrations must be due to the lack of CbbLS-1 and to the simultaneous reduction of CbbM, thus indicating that CbbLS-2 is inappropriate for optimum growth under high CO2 conditions.
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DISCUSSION |
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We introduced genetic engineering in H. marinus and constructed knockout mutants of the cbbR regulatory genes. CbbR1 seems to be essential for the transcription of cbbLS-1, because the cbbR1 mutant strain dR1 lost the ability to synthesize CbbLS-1 under all CO2 conditions. The expression of the other RubisCOs was not influenced by the mutation (Fig. 4). Strain dR1 grew at the same rate as the wild-type strain under all CO2 conditions, indicating that under all CO2 conditions, CbbLS-1 does not have a crucial role for growth. The cbbRm mutant strain dRm also showed the same growth profiles as those of the wild-type strain. No activation of CbbM expression at higher CO2 concentrations (
2 %) was observed in the mutant. However, a significant increase in CbbLS-1 was observed under these conditions, suggesting that the increase in CbbLS-1 functionally compensated for the decrease in CbbM. A similar compensatory effect has also been observed in the RubisCO gene mutants of R. sphaeroides and R. capsulatus (Gibson et al., 1991
; Paoli et al., 1998
). The RNase protection assays revealed that under all conditions the expression level of cbbRm did not change drastically in strain MH-110 (Fig. 6
), indicating that the activation of cbbM expression at higher CO2 concentrations (
2 %) required some factor that acts cooperatively with CbbRm. The cbbR1 cbbRm double knockout strain ddR showed poor growth at higher CO2 concentrations (
2 %) (Fig. 5
). The expression levels of CbbLS-1 and CbbM were decreased in the strain (Fig. 4
). Interestingly, the expression of CbbLS-2 was observed at higher CO2 concentrations (
2 %) in strain ddR. These results indicated that the high-level expression of CbbLS-1 or CbbM is necessary for optimal growth at higher CO2 concentrations, and that CbbLS-2 does not functionally compensate for the lack of CbbLS-1 and CbbM.
Disruption of cbbRm led to the high-level expression of CbbLS-1 at higher CO2 concentrations, and the disruption of both cbbR1 and cbbRm caused the expression of CbbLS-2 at higher CO2 concentrations. These results indicated that H. marinus has a hierarchical interactive regulatory mechanism among the three RubisCOs. R. capsulatus possesses two cbb operons, and the expression of each operon is activated by the cognate CbbR regulators (Vichivanives et al., 2000). An interactive regulation of the two cbb operons by the cross-reactions of the CbbR regulators is indicated in R. capsulatus. The cross-reaction of the CbbR regulators might be also operative in the interactive regulation of RubisCOs in H. marinus. CbbRm might act as a repressor for the cbbR1 promoter, and both CbbR1 and CbbRm might act as repressors for the cbbLS-2 promoter. However, because the affinity of the CbbR regulators to the non-cognate promoters was lower than that to the cognate promoters, the cross-reaction by the CbbRs seems to be insufficient to explain the interactive regulation. Disruption of cbbR1 or cbbRm caused a significant decrease in their cognate RubisCO enzymes, which might change the flux of the CBB cycle. Another possible explanation for the cross-regulation is that an accumulation of certain intermediates or a change in certain cellular states caused by the loss of CbbLS-1 and/or CbbM is the trigger for the induction of another RubisCO. It has been reported that the DNA binding of CbbR is altered in the presence of certain metabolites in other bacteria (van Keulen et al., 1998
, 2003
; Grzeszik et al., 2000
; Terazono et al., 2001
; Dubbs et al., 2004
). In R. capsulatus, the binding of both CbbRI and CbbRII to their cognate promoter was enhanced in the presence of certain metabolites formed by the CBB cycle, as well as other intermediates, including ribulose-1,5-bisphosphate, 3-phosphoglycerate and PEP (Dubbs et al., 2004
). These results indicated that the CBB cycle-related metabolites act as the signals for the expression of RubisCO enzymes, thus supporting the explanation mentioned above.
The growth and expression of cbbLS-2 at lower CO2 concentrations (0·15 %) was not affected by the disruption of cbbR1 and/or cbbRm, suggesting that growth at the lower CO2 concentrations might primarily be supported by CbbLS-2. The cbbLS-2 genes are followed by genes encoding carboxysome, a polyhedral inclusion body that concentrates CO2 for RubisCO, which is included in the body. Carboxysome, which is conserved in all cyanobacteria and some chemoautotrophic bacteria, is a part of the CO2-concentrating mechanism (CCM), which is induced by CO2 limitation and has been studied intensively in cyanobacteria and thiobacilli (Badger & Price, 2003
; Cannon et al., 2003
). Because RubisCO has a low affinity for CO2 and a slow catalytic rate, it requires the CCM under low-CO2 conditions. Since the synthesis of carboxysome is clearly an energetic and metabolic burden for the cell, the expression of the cbbLS-2 operon containing carboxysome genes must be strictly regulated. The threshold CO2 concentration for the induction of cbbLS-2 and carboxysome is somewhere between 2 and 0·15 %. However, the CO2 molecule is not likely to be the direct effector for the expression of cbbLS-2, because cbbLS-2 was expressed even at higher CO2 concentrations in strain ddR. Change in the expression levels at concentrations between 2 and 0·15 % was also found in the cbbR1 transcript and the CbbM protein. Their regulation was opposite to that of cbbLS-2. These results suggest that drastic changes in the cellular states that affect the expression of the cbb genes occur at CO2 concentrations between 2 and 0·15 %. Identification of the sensing signals for the CbbRs may contribute to a more complete understanding of the network responsible for the regulation of RubisCO expression in H. marinus.
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Received 19 March 2005;
revised 8 July 2005;
accepted 3 August 2005.
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