The role of two CbbRs in the transcriptional regulation of three ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Hydrogenovibrio marinus strain MH-110

Koichi Toyoda, Yoichi Yoshizawa, Hiroyuki Arai, Masaharu Ishii and Yasuo Igarashi

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hydrogenovibrio marinus MH-110 possesses three different sets of genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): two form I (cbbLS-1 and cbbLS-2) and one form II (cbbM). We have previously shown that the expression of these RubisCO genes is dependent on the ambient CO2 concentration. LysR-type transcriptional regulators, designated CbbR1 and CbbRm, are encoded upstream of the cbbLS-1 and cbbM genes, respectively. In this study, we revealed by gel shift assay that CbbR1 and CbbRm bind with higher affinity to the promoter regions of cbbLS-1 and cbbM, respectively, and with lower affinity to the other RubisCO gene promoters. The expression patterns of the three RubisCOs in the cbbR1 and the cbbRm gene mutants showed that CbbR1 and CbbRm were required to activate the expression of cbbLS-1 and cbbM, respectively, and that neither CbbR1 nor CbbRm was required for the expression of cbbLS-2. The expression of cbbLS-1 was significantly enhanced under high-CO2 conditions in the cbbRm mutant, in which the expression of cbbM was decreased. Although cbbLS-2 was not expressed under high-CO2 conditions in the wild-type strain or the single cbbR mutants, the expression of cbbLS-2 was observed in the cbbR1 cbbRm double mutant, in which the expression of both cbbLS-1 and cbbM was decreased. These results indicate that there is an interactive regulation among the three RubisCO genes.


Abbreviations: CBB cycle, Calvin–Benson–Bassham cycle; LTTR, LysR-type transcriptional regulator; PEP, phosphoenolpyruvate; RubisCO, D-ribulose 1,5-bisphosphate carboxylase/oxygenase


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Calvin–Benson–Bassham (CBB) cycle plays the most important role in assimilating CO2 into organic carbon on earth, since all plants and cyanobacteria use this cycle to fix CO2, as do many autotrophic bacteria. D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39), one of two unique enzymes in the pathway, catalyses the actual CO2 fixation reaction. Four forms of RubisCO have been identified to date: form I, encoded by cbbLS, is composed of eight large (CbbL) and eight small subunits (CbbS) and is found in all plants and cyanobacteria, and in some proteobacteria; form II, encoded by cbbM, consists of only large subunits of varying numbers, depending on the organism examined; form III, found in archaea, is also composed of large subunits (Kitano et al., 2001; Finn & Tabita, 2003); and form IV, reported recently, is referred to as RubisCO-like protein (RLP) (Hanson & Tabita, 2001; Ashida et al., 2003). RLP is incapable of catalysing the fixation of CO2, but is likely to be involved in the regulation of sulfur metabolism in Bacillus subtilis (Ashida et al., 2003). The distribution and function of each form of RubisCO mentioned above indicate that form I and form II are the principal forms of RubisCO that assimilate CO2 in the atmosphere.

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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Fig. 1. The organization of three RubisCO operons of H. marinus strain MH-110. The arrows indicate the size and direction of the genes. cbbL and cbbS encode large and small subunits of form I RubisCO, respectively. cbbM encodes form II RubisCO. cbbR encodes the LysR-type transcriptional regulator (LTTR). cbbQ and cbbO encode proteins thought to be involved in the post-translational activation of RubisCO. cso genes encode carboxysome proteins. bfr encodes bacterioferritin. can encodes carbonic anhydrase.

 
CbbR, one of the LysR-type transcriptional regulators (LTTRs), has been found in many photo- and chemoautotrophic bacteria, and has been shown to be required to activate expression of RubisCO (Windhövel & Bowien, 1991; Falcone & Tabita, 1993; Gibson & Tabita, 1993; van den Bergh et al., 1993; Paoli et al., 1998; Dubbs et al., 2000, 2004; Vichivanives et al., 2000). Although the cbbR genes are located upstream of genes encoding RubisCO in a divergent orientation in most bacteria, the number of cbbR genes is not equivalent to that of genes for RubisCO (Windhövel & Bowien, 1991; Gibson & Tabita, 1993; Meijer et al., 1996; Kusian & Bowien, 1997; Smith & Tabita, 2002; Dubbs & Tabita, 2003). For example, a single cbbR gene product regulates two cbb operons in Rhodobacter sphaeroides (Gibson & Tabita, 1993; Smith & Tabita, 2002; Dubbs & Tabita, 2003). In H. marinus, the cbbR genes were found upstream of cbbLS-1 and cbbM, and were designated cbbR1 and cbbRm, respectively; however, no regulatory genes were found upstream of cbbLS-2 (Fig. 1) (Yoshizawa et al., 2004).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media, and growth conditions.
The plasmids and bacterial strains used in this study are listed in Table 1. Escherichia coli strains (Yanisch-Perron et al., 1985) were grown aerobically in Luria–Bertani (LB) medium at 37 °C. H. marinus was grown in 500 ml of inorganic medium (Yoshizawa et al., 2004) bubbled continuously with a gas mixture (H2/O2/CO2) at the rate of 500 ml min–1 in a 1 l fermenter (Able, Tokyo, Japan) at 37 °C. The CO2 concentration in the gas mixture was adjusted by changing the ratio of CO2 in the gas mixture, as described previously (Yoshizawa et al., 2004), i.e. the 2 % CO2 concentration was achieved with a gas mixture (H2 : O2 : CO2=83 : 15 : 2), with the exception that the gas containing 0.03 % CO2 was a mixture of 20 % H2/80 % air. The antibiotic concentrations used for H. marinus strains were as follows: kanamycin (Km) 15 µg ml–1, tetracycline (Tc) 2 µg ml–1, gentamicin (Gm) 10 µg ml–1, streptomycin (Sm) 10 µg ml–1. For E. coli, the antibiotic concentrations were as follows: ampicillin, 100 µg ml–1; Km, 30 µg ml–1; Tc, 12·5 µg ml–1; Gm, 10 µg ml–1; Sm, 15 µg ml–1.


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Table 1. Bacterial strains and plasmids used in this study

 
Expression of the two H. marinus CbbRs in E. coli.
The two CbbRs of H. marinus, CbbR1 and CbbRm, were overexpressed in E. coli for purification. The coding region of the cbbR1 gene was amplified by PCR from the total genomic DNA of H. marinus as template, using primers 5'-ATAGAGCATACATATGCAAAATTTAC-3' and 5'-CTTTTTACCCGGGCTATCTTGTATC-3' for cbbR1. The PCR reaction consisted of 500 pmol of each of the oligonucleotide primers, 500 ng DNA template and 2·5 units Ex Taq polymerase (TaKaRa). The amplified fragment containing cbbR1 was ligated with the expression vector pTYB12 using the appropriate restriction sites, resulting in pTYBR1. The constructed plasmid was designed to encode fusion proteins of CbbR1 with an intein and a chitin-binding domain (CBD). The recombinant fusion protein was expressed in E. coli strain ER2566 and purified according to the manufacturer's instructions (New England BioLabs). In brief, the cell lysate was loaded on a chitin column. The recombinant CbbR1 was excised from the intein–CBD fusion complex on the column by an auto-cleavage reaction from the intein moiety at 4 °C in the presence of DTT.

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 orf90–cbbRm 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 orf90–cbbRm. 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 orf90–cbbRm, 227 bp. The fragments were labelled by either filling the recessed end with [{alpha}-32P]dATP (Amersham Pharmacia) using Klenow enzyme or phosphorylating the end of the fragments with [{gamma}-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 [{alpha}-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 [{gamma}-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 ml–1 poly(dI-dC).poly(dI-dC) (Amersham Pharmacia) and 50 µg ml–1 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 cm–1. 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 PvuII–StuI 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 {lambda}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{Omega}aac (Blondelet-Rouault et al., 1997) was inserted into the unique BalI site in the cbbR1 gene on pUCR1. The EcoRI–XbaI fragment containing the disrupted cbbR1 gene on the constructed plasmid was excised and cloned into pJP5608, resulting in pJPGR1. E. coli strain S17-1 {lambda}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 [{alpha}-32P]UTP [800 Ci mmol–1 (29600 GBq mmol–1), 10 mCi ml–1 (370 MBq mmol–1); 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 [{gamma}-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 µl–1) 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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METHODS
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Binding of CbbR1 and CbbRm to the promoter regions of the cbb genes
Given that CbbR1 and CbbRm belong to the LTTR family, these CbbRs could bind to the promoter regions of RubisCO genes and activate their expression in H. marinus MH-110. To examine the ability of both CbbR1 and CbbRm to bind to these regions, gel shift assays were performed. CbbR1 was expressed in E. coli as a fusion protein with an intein containing a chitin-binding domain that was removed by treatment with DTT on a chitin column during purification. CbbRm was expressed as a His-tagged protein in E. coli and was purified using Ni2+ affinity chromatography. The DNA fragments containing the promoter regions of cbbLS-1, cbbLS-2 and cbbM were end-labelled with 32P and incubated with CbbR1 or CbbRm. The gel shift assays revealed that CbbR1 and CbbRm were able to bind to the promoter regions of cbbLS-1 and cbbM, respectively (Fig. 2). Excess amounts of the unlabelled fragment eliminated the shifted bands, indicating that the band-shift by CbbR1 or CbbRm was sequence-specific. The results also indicated that CbbR1 and CbbRm bound to the promoter regions of other RubisCO operons with lower affinity. However, excess amounts of the unlabelled fragments of the other promoters did not completely inhibit the binding of CbbR1 to the cbbLS-1 promoter or of CbbRm to the cbbM promoter. These results indicated that CbbR1 and CbbRm primarily regulate the expression of cbbLS-1 and cbbM, respectively. CbbR1 gave two shifted bands for the cbbLS-1 promoter and the ratio of the upper shifted band to the lower one was increased with an increase in protein concentrations. CbbRm also gave two shifted bands for the cbbM promoter at higher protein concentrations. It has been shown that CbbR binds to the target promoter with two subsites and forms a dimer of dimers on the promoter in some bacteria (Kusano & Sugawara, 1993; Kusian & Bowien, 1995; van Keulen et al., 1998, 2003). Thus, CbbR1 and CbbRm of H. marinus probably bind to each target promoter as a dimer of dimers.



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Fig. 2. Binding of CbbR1 and CbbRm to the promoter region of the cbb genes. (a) Gel shift assays using CbbR1 with DNA fragments containing the promoter regions of cbbLS-1, cbbLS-2, cbbM and cbbRm. The labelled fragments were incubated with purified CbbR1. The protein concentrations used for the cbbLS-2, cbbM and cbbRm promoter regions were as follows (in ng per 20 µl reaction mixture): 0 (lane 1), 10 (lane 2), 50 (lane 3), 250 (lane 4). For the cbbLS-1 promoter region, the protein concentrations were as follows (in ng per 20 µl reaction mixture): 0 (lane 1), 5 (lane 2), 10 (lane 3), 20 (lanes 4, 7–9), 50 (lane 5), 100 (lane 6). A 50-fold molar excess of unlabelled fragment of the cbbLS-1 (lane 7), cbbLS-2 (lane 8) or cbbM (lane 9) promoter was added to the mixture prior to the addition of labelled fragments. (b) Gel shift assays using CbbRm with DNA fragments containing the promoter regions of cbbLS-1, cbbLS-2, cbbM and cbbRm. The labelled fragment was incubated with purified CbbRm. The protein concentrations used for cbbLS-1, cbbLS-2 and cbbRm promoter regions were as follows (in ng per 20 µl reaction mixture): 0 (lane 1), 10 (lane 2), 50 (lane 3), 250 (lane 4). For the cbbM promoter region, the protein concentrations were as follows (in ng per 20 µl reaction mixture): 0 (lane 1), 5 (lane 2), 10 (lane 3), 20 (lanes 4, 7–9), 50 (lane 5), 100 (lane 6). A 50-fold molar excess of unlabelled fragment of the cbbLS-1 (lane 7), cbbLS-2 (lane 8) or cbbM (lane 9) promoter was added to the mixture prior to the addition of labelled fragments. The open and filled arrow boxes indicate free DNA probe and DNA–protein complex, respectively. Asterisks indicate contaminated non-specific bands.

 
To identify and analyse the promoter regions of each cbb operon and of the two cbbR genes, the transcription start sites of each cbb gene were determined (Fig. 3). The transcription start sites of the cbbLS-1 and cbbLS-2 operons were located at positions 103 bp and 37 bp, respectively, upstream of the first gene of each operon. cbbM had two transcriptional start points at positions –129 bp and –123 bp. The transcription start site of cbbR1 was located at position –56 bp. The size of a product of the primer extension experiment using oligonucleotide cbbRm-1 exceeded 300 nt (data not shown). An ORF search revealed that a 270 bp gene, designated orf90, was located upstream of cbbRm in the same direction as cbbRm. A primer extension experiment using cbbRm-2, which was specific to orf90, revealed that the transcription start sites of the cbbRm operon were located at positions 41 and 42 bp upstream of orf90. The physiological role of the orf90 gene product was unclear, because no homologous sequence could be found in public databases.



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Fig. 3. Mapping of the transcription initiation sites of the cbbLS-1, cbbLS-2, cbbM, cbbR1 and cbbRm promoters. Primer extension experiments were performed using [5'-32P]-labelled oligonucleotides. Total RNA extracted from cells grown under 2 % CO2 was used to identify (a) the cbbLS-1, (c) cbbM, (d) cbbR1 and (e) cbbRm transcriptional initiation sites. Total RNA extracted from cells grown under 0·15 % CO2 was used to identify (b) the cbbLS-2 transcriptional initiation site. Arrows indicate the transcription initiation sites. Lanes A, C, G and T show the sequencing reaction using the same primer. Lane RT shows the primer extension product.

 
Characterization of cbbR1 and cbbRm mutant strains
To examine the physiological roles of CbbR1 and CbbRm, their genes were disrupted by homologous recombination. The cbbR1 gene disruptant, strain dR1, showed a similar growth pattern to that of the wild-type strain MH-110 under all CO2 conditions examined (data not shown). Western immunoblot analyses using antibodies against each RubisCO revealed that CbbLS-1 was not expressed in strain dR1 under any of the CO2 conditions (Fig. 4b). The expression levels of CbbM and CbbLS-2 were not affected by the mutation. These results indicated that the expression of CbbM alone is enough for growth at higher CO2 concentrations. Transformation of strain dR1 with the recombinant cbbR1 gene by pCR1 restored the expression of CbbLS-1 (data not shown). CbbLS-1 was not expressed at 15 % CO2 concentration in the wild-type strain MH-110 in a previous study (Yoshizawa et al., 2004), but slight expression was observed in this work (Fig. 4a). The reason for the difference was not certain, but a small difference in the culture conditions seemed to affect the expression of CbbLS-1 at this CO2 concentration.



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Fig. 4. Western immunoblots of (a) H. marinus wild-type strain MH-110, (b) cbbR1 strain dR1, (c) cbbRm strain dRm and (d) cbbR1 cbbRm double mutant strain ddR using antibodies for CbbS-1 (left), CbbS-2 (centre) and CbbM (right). Cell-free extracts (approx. 10 µg total protein) of H. marinus strains grown at 15 % (lane 1), 2 % (lane 2), 0·15 % (lane 3) and 0·03 % (lane 4) CO2 concentrations were used. The samples from the wild-type strain and each mutant were analysed on the same gel to allow a quantitative comparison. Panel (a) shows a representative result from each analysis.

 
The cbbRm gene disruptant, strain dRm, also grew at the same rate as MH-110 under all CO2 conditions examined (data not shown). Western immunoblot analyses revealed that the expression levels of cbbM at CO2 concentrations above 2 % were significantly decreased in strain dRm (Fig. 4c). However, CbbM expression was not completely abolished by the disruption of cbbRm, indicating that the transcription of cbbM was basically constitutive and not totally dependent on CbbRm. In contrast, a significant increase in CbbLS-1 was found at CO2 concentrations above 2 % in this mutant. These results indicated that the increased expression of CbbLS-1 complemented the decrease in CbbM and supported growth at higher CO2 concentrations. CbbLS-1 was also expressed at 0·15 % CO2 concentration in strain dRm, although it was not expressed in MH-110. The expression level of CbbLS-1 at 0·03 % CO2 concentration was also higher in strain dRm than in MH-110. The disruption of the cbbRm gene had no effect on the expression of cbbLS-2 under any of the CO2 conditions. The expression patterns of CbbM and CbbLS-1 in dRm were the same as those of MH-110 when transformed with the recombinant cbbRm gene by pCRm (data not shown).

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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Fig. 5. Growth profiles of the wild-type and the cbbR1 cbbRm mutant ddR. The wild-type and strain ddR were cultivated under four CO2 conditions (percentage CO2 concentrations shown). {bullet}, Wild-type; {blacktriangledown}, ddR. The data are representative of duplicate experiments.

 
Expression analyses of cbbR1 and cbbRm
To investigate the relationship between the expression patterns of the three RubisCO genes in H. marinus at various CO2 concentrations and those of cbbR genes, the expression of cbbR1 and cbbRm was analysed by RNase protection assays. Riboprobes containing the last 107 and 110 codons of CbbR1 and CbbRm, respectively, were used to detect the 3' ends of cbbR1 and cbbRm transcripts, but not to detect the 5' ends of their truncated transcripts. The assays revealed that the amount of the cbbR1 transcripts increased when the CO2 concentration was higher than 2 % (Fig. 6), indicating that cbbR1 is regulated by another regulator that is able to respond to changes in the CO2 concentration. CbbR1 is divergently encoded upstream of cbbLS-1. The gel shift assay showed that CbbR1 bound to the overlapping promoter region between cbbLS-1 and cbbR1, suggesting that the expression of CbbR1 is subjected to autoregulation. Negative autoregulation has also been demonstrated for the LTTR including CbbR (Schell, 1993; Vichivanives et al., 2000; van Keulen et al., 2003). The amount of the cbbRm transcripts did not vary significantly under any of the CO2 conditions. CbbRm is encoded upstream of cbbM in the same direction, and the gel shift assay showed that neither CbbR1 nor CbbRm bound to the promoter region of cbbRm (Fig. 2), indicating that the expression of CbbRm is not regulated by either of the two CbbRs.



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Fig. 6. Ribonuclease protection analyses of the cbbR1 and cbbRm transcripts in wild-type strain MH-110 under different CO2 conditions. (a) The cbbR1 transcript was observed using a 411 bp riboprobe containing the last 107 codons of cbbR1. The expected size of the RNase-protected fragments of this cbbR1 transcript was 321 bp. (b) The cbbRm transcript was detected with a 440 bp riboprobe containing the last 110 codons of cbbRm. The expected size of the RNase-protected fragments of this transcript was 330 bp. The cbbR1 or cbbRm probe was hybridized to yeast RNA in the absence (lane 1) or presence (lane 2) of added RNase A/T1. The probes were hybridized with RNA preparations from cells grown at 15 % (lane 3), 2 % (lane 4), 0·15 % (lane 5) or 0·03 % (lane 6) CO2. Ten (a) and 20 µg (b) total RNA were used to detect the cbbR1 and cbbRm transcripts, respectively. The open and filled arrowheads indicate the size of undigested probe and a protected band, respectively. Results are representative of three independent experiments with similar results.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In a previous report, we demonstrated that the ambient CO2 concentration affects the expression of three RubisCOs in H. marinus (Yoshizawa et al., 2004). The present study clarified in part the complicated regulatory mechanism of RubisCO gene expression by the analysis of the roles played by two LTTRs, CbbR1 and CbbRm. The results of the gel shift assays demonstrated that CbbR1 and CbbRm of H. marinus bound with higher affinity to the promoter regions of cbbLS-1 and cbbM, respectively (Fig. 2), indicating that these genes are mainly under the control of their adjacently encoded regulators. We also found that CbbR1 and CbbRm bound to the promoter regions of the other RubisCO operons. However, the interaction was weak compared to that observed between CbbR1 and the cbbLS-1 promoter or CbbRm and the cbbM promoter, suggesting that the binding of CbbR1 and CbbRm to the non-cognate promoter has a minor role in vivo.

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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