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Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre

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Abstract

A new gene expression system was developed in Rhodobacter sphaeroides, replacing a pRK415-based system used previously. The broad host-range IPTG-inducible plasmid pIND4 was used to create the plasmid pIND4-RC1 for expression of the puhA and pufQBALMX genes, encoding the reaction centre (RC) and light-harvesting complex 1 (LH1) proteins. The strain R. sphaeroides ΔRCLH was used to make a knockout of the rshI restriction endonuclease gene, enabling electroporation of DNA into the bacterium; a subsequent knockout of ppsR was made, creating the strain R. sphaeroides RCx lacking this oxygen-sensing repressor of the photosynthesis gene cluster. Using pIND4-RC1, LH1 levels were increased by a factor of about 8 over pRS1 per cell in cultures grown semi-aerobically. In addition, the ppsR knockout allowed for photosynthetic pigment–protein complex synthesis in the presence of high concentrations of molecular oxygen; here, LH1 levels per cell increased by 20 % when grown under high aeration conditions. A new medium (called RLB) is the E. coli medium LB supplemented with MgCl2 and CaCl2, which was found to increase growth rates and final cell culture densities, with an increase of 30 % of LH1 per cell detected in R. sphaeroides RCx(pIND4-RC1) grown in RLB versus LB medium. Furthermore, cell density was about three times greater in RLB compared to semi-aerobic conditions. The combination of all the modifications resulted in an increase of LH1 and RC per mL of culture volume by approximately 35-fold, and a decrease in the length of culture incubation time from about 5 days to ~36 h.

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References

  • Abresch EC, Axelrod HLA, Beatty JT, Johnson JA, Nechushtai R, Paddock ML (2005) Characterization of a highly purified, fully active, crystallizable RC–LH1–PufX core complex from Rhodobacter sphaeroides. Photosynth Res 86:61–70

    Article  CAS  PubMed  Google Scholar 

  • Beatty JT, Gest H (1981) Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol 129:335–340

    Article  CAS  Google Scholar 

  • Brimacombe CA, Stevens A, Jun D, Mercer R, Lang AS, Beatty JT (2013) Quorum-sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA). Mol Microbiol 87:802–817

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen X-Y, Yurkov V, Paddock M, Okamura M, Beatty JT (1998) A puhA gene deletion and plasmid complementation system for facile site directed mutagenesis studies of the reaction center H protein of Rhodobacter sphaeroides. Photosynth Res 55:369–373

    Article  CAS  Google Scholar 

  • Clayton RK (2002) Research on photosynthetic reaction centers from 1932 to 1987. Photosynth Res 73:63–71

    Article  CAS  PubMed  Google Scholar 

  • Clayton RK, Smith C (1960) Rhodopseudomonas sphaeroides: high catalase and blue–green double mutants. Biochem Biophys Res Commun 3:143–145

    Article  CAS  PubMed  Google Scholar 

  • Clayton RK, Wang RT (1971) Photochemical reaction centers from Rhodopseudomonas sphaeroides. Methods Enzymol 23:696–704

    Article  Google Scholar 

  • Donohue TJ, Kaplan S (1991) Genetic techniques in Rhodospirillaceae. Methods Enzymol 204:459–485

    Article  CAS  PubMed  Google Scholar 

  • Elsen S, Jaubert M, Pignol D, Giraud E (2005) PpsR: a multifaceted regulator of photosynthesis gene expression in purple bacteria. Mol Microbiol 57:17–26

    Article  CAS  PubMed  Google Scholar 

  • Feher G (1971) Some chemical and physical properties of a bacterial reaction center particle and its primary photochemical reactants. Photochem Photobiol 14:373–388

    Article  CAS  PubMed  Google Scholar 

  • Feher G (1998) Three decades of research in bacterial photosynthesis and the road leading to it: a personal account. Photosynth Res 55:1–40

    Article  Google Scholar 

  • Goldsmith JO, Boxer SG (1996) Rapid isolation of bacterial photosynthetic reaction centers with an engineered polyhistidine tag. Biochim Biophys Acta 1276:171–175

    Article  Google Scholar 

  • Gomelsky M, Horne IM, Lee HJ, Pemberton J, McEwan A, Kaplan S (2000) Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression. J Bacteriol 182:2253–2261

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Govindjee, Beatty JT, Gest H (2003) Celebrating the millennium—historical highlights of photosynthesis research, Part 2. Photosynth Res 76:1–11

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  CAS  PubMed  Google Scholar 

  • Ind AC, Porter SL, Brown MT, Byles ED, de Beyer JA, Godfrey SA, Armitage JP (2009) Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans. Appl Environ Microbiol 75:6613–6615

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Inui M, Nakata K, Roh JH, Vertes AA, Yukawa H (2003) Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobacter blasticus. Appl Environ Microbiol 69:725–733

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jaschke PR, Saer RG, Noll S, Beatty JT (2011) Modification of the genome of Rhodobacter sphaeroides and construction of synthetic operons. Methods Enzymol 497:519–538

    Article  CAS  PubMed  Google Scholar 

  • Jones MR, Fowler GJS, Gibson LCD, Grief GG, Olsen JD, Crielaard W, Hunter CN (1992a) Mutants of Rhodobacter sphaeroides lacking one or more pigment–protein complexes and complementation with reaction-centre, LH1, and LH2 genes. Mol Microbiol 6:1173–1184

    Article  CAS  PubMed  Google Scholar 

  • Jones MR, Visschers RW, Van Grondelle R, Hunter CN (1992b) Construction and characterization of a mutant of Rhodobacter sphaeroides with the reaction center as the sole pigment–protein complex. Biochemistry 31:4458–4465

    Article  CAS  PubMed  Google Scholar 

  • Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70:191–197

    Article  CAS  PubMed  Google Scholar 

  • Kondo M, Iida K, Dewa T, Tanaka H, Ogawa T, Nagashima S, Nagashima KVP, Shimada K, Hashimoto H, Gardiner AT, Cogdell R, Nango M (2012) Photocurrent and electronic activities of oriented-His-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode. Biomacromolecules 13:432–438

    Article  CAS  PubMed  Google Scholar 

  • Lebedev N, Trammell SA, Spano A, Lukashev E, Griva I, Schnur J (2006) Conductive wiring of immobilized photosynthetic reaction center to electrode by cytochrome c. J Am Chem Soc 128:12044–12045

    Article  CAS  PubMed  Google Scholar 

  • Mahmoudzadeh A, Saer R, Jun D, Mirvakili SM, Takshi A, Iranpour B, Ouellet E, Lagally ET, Madden JDW, Beatty JT (2011) Photocurrent generation by direct electron transfer using photosynthetic reaction centres. Smart Mater Struct 20:1–6

    Google Scholar 

  • Martin E, Baldansuren A, Lin T-J, Samoilova RI, Wraight CA, Dikanov SA, O’Malley PJ (2012) Hydrogen bonding between the QB site ubisemiquinone and Ser-L223 in the bacterial reaction center: a combined spectroscopic and computational perspective. Biochemistry 51:9086–9093

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Norris V, Chen M, Goldberg M, Voskuil J, McGurk G, Holland B (1991) Calcium in bacteria: a solution to which problem? Mol Microbiol 5:775–778

    Article  CAS  PubMed  Google Scholar 

  • Paddock ML, Rongey SH, Feher G, Okamura MY (1989) Pathway of proton transfer in bacterial reaction centers: replacement of glutamic acid 212 in the L subunit by glutamine inhibits quinone (secondary acceptor) turnover. Proc Natl Acad Sci USA 86:6602–6606

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pokkuluri PR, Laible PD, Deng Y-L, Wong TN, Hanson DK, Schiffer M (2002) The structure of a mutant photosynthetic reaction center shows unexpected changes in main chain orientations and quinone position. Biochemistry 41:5998–6007

    Article  CAS  PubMed  Google Scholar 

  • Roh JH, Smith WE, Kaplan S (2004) Effects of oxygen and light intensity on transcriptome expression in Rhodobacter sphaeroides 2.4.1. J Biol Chem 279:9146–9155

    Article  CAS  PubMed  Google Scholar 

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol 1:784–791

    Article  CAS  Google Scholar 

  • Sistrom WR (1960) A requirement for sodium in the growth of Rhodopseudomonas sphaeroides. Microbiology 22:778–785

    CAS  Google Scholar 

  • Sistrom WR (1962) The kinetics of the synthesis of photopigments in Rhodopseudomonas sphaeroides. J Gen Microbiol 28:607–616

    Article  CAS  PubMed  Google Scholar 

  • Tehrani A, Beatty JT (2004) Effects of precise deletions in Rhodobacter sphaeroides reaction center genes on steady-state levels of reaction center proteins: a revised model for reaction center assembly. Photosynth Res 79:101–108

    Article  CAS  PubMed  Google Scholar 

  • Trammell SA, Wang L, Zullo JM, Shashidhar R, Lebedev N (2004) Orientated binding of photosynthetic reaction centers on gold using Ni–NTA self-assembled monolayers. Biosens Bioelectron 19:1649–1655

    Article  CAS  PubMed  Google Scholar 

  • Yaghoubi H, Li Z, Jun D, Saer R, Slota JE, Beerbom M, Schlaf R, Madden JD, Beatty JT, Takshi A (2012) The role of gold-adsorbed photosynthetic reaction centers and redox mediators in the charge transfer and photocurrent generation in a bio-photoelectrochemical cell. J Phys Chem C 116:24868–24877

    Article  CAS  Google Scholar 

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Acknowledgments

We thank the Canadian Natural Sciences and Engineering Research Council (NSERC) for Discovery Grants and a Strategic Grant awarded to JTB and JDM.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada

    D. Jun, R. G. Saer & J. T. Beatty

  2. Department of Electrical and Computer Engineering, The University of British Columbia, Vancouver, BC, Canada

    J. D. Madden

Authors
  1. D. Jun
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  2. R. G. Saer
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  3. J. D. Madden
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  4. J. T. Beatty
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Correspondence to J. T. Beatty.

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Jun, D., Saer, R.G., Madden, J.D. et al. Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre. Photosynth Res 120, 197–205 (2014). https://doi.org/10.1007/s11120-013-9866-6

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  • DOI: https://doi.org/10.1007/s11120-013-9866-6

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