Abstract
We report here the construction of a homozygous recA460::cam insertion mutant of Synechocystis sp. PCC 6803 that may be useful for plant molecular genetics by providing a plant like host free of interference from homologous recombination. The homozygous recA460::cam mutant is highly sensitive to UVC under both photoreactivating and nonphotoreactivating conditions compared to the wild type (WT). The liquid culture of the mutant growing in ~800 lx accumulates nonviable cells to the tune of 86% as estimated by colony counts on plates incubated at the same temperature and light intensity. The generation time of recA mutant in standard light intensity (2,500 lx) increases to 50 h compared to 28 h in lower light intensity (~800 lx) that was used for selection, thus explaining the earlier failures to obtain a homozygous recA mutant. The WT, in contrast, grows at faster rate (23 h generation time) in standard light intensity compared to that at ~800 lx (26 h). The Synechocystis RecA protein supports homologous recombination during conjugation in recA — mutant of Escherichia coli, but not the SOS response as measured by UV sensitivity. It is suggested that using this homozygous recA460::cam mutant, investigations can now be extended to dissect the network of DNA repair pathways involved in housekeeping activities that may be more active in cyanobacteria than in heterotrophs. Using this mutant for the first time we provide a genetic evidence of a mechanism independent of RecA that causes enhanced UVC resistance on light to dark transition.
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Abbreviations
- UV:
-
Ultraviolet radiation
- UVC:
-
Ultraviolet radiation (254 nm)
- Cam:
-
chloramphenicol resistance cassette
References
Allen MM (1968) Simple conditions for growth of unicellular blue-green algae on plates. J Phycol 4: 1–4
Bhattacharjee SK (1977) Unstable protein mediated ultraviolet light resistance in Anacystis nidulans. Nature 269:82–83
Bhattacharjee SK (1989) Sensitivity to Far-UV light as a parameter for the study of in vivo regulation of development of PSII particles in cyanobacteria: An analytical model. In: Singhal GS, Barber J, Dilley RA, Govindjee R, Haselkorn, Mohanty P (eds) Photosynthesis molecular biology and bioenergetics. Narosa Publishing House, New Delhi pp 267–281
Bhattacharjee SK, David KAV (1977) Unusual resistance in the dark phase of blue-green bacterium Anacystis nidulans. Nature 265:183–184
Bialkowski K, Olinski R (1999) Oxidative damage to plant DNA in relation to growth condition. Acta Biochim Pol 46:43–49
Britt AB (1999) Molecular genetics of DNA repair in higher plants. Trends Plan Sci 4:20–25
Buchholz G, Frohmeyer H, Hohn B (2000) UV-damage mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation. Proc Natl Acad Sci USA 97:13425–13429
Capaldo-Kimball FN, Barour SD (1971). Involvement of recombination genes in growth and viability of Escherichia coli K-12. J Bacteriol 106:204–212
Capaldo FN, Ramsey G, Barbour SD (1974) Analysis of the growth of recombination-deficient strains of Escherichia coli K-12. J Bacteriol 118:242–249
Clark AJ, Margulis AD (1965) Isolation and charecterization of recombination-deficient mutants of Escherichia coli K 12. Proc Natl Acad Sci.USA.53: 451–459
Clark AJ, Chamberlin M. Boyce RP, Howard-Flanders P (1966) Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli. J Mol Biol 19:442–454
Castenholz RW, Garcia-Pichel F (2000) Cyanobacterial response to UV radiation. In: Whitton BA Potts M (eds) The ecology of cyanobacteria. Kluwer, Dordhecht pp 591–611
Cooper AJ, Kalinowski AP, Shoemaker NB, Slayers AA (1997) Construction and characterization of Bacteroides thetaiotaomicron recA mutant: Transfer of Bacteroides integrated congugative elements is RecA independent. J Bacteriol 179:6221–6227
Courcelle J, Hanawalt PC (2001) Participation of protein in rescue of arrested replication fork in UV irradiated Escherichia coli need not involve recombination. Proc Natl Acad Sci USA 98:8196–8202
Courcelle J, Hanawalt PC (2003) RecA-dependent recovery of arrested DNA replication forks. Ann Rev Genet 37:611–646
Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ (2000) The importance of repairing stalled replication forks. Nature 404:37–41
David KAV, Rane SS, Mathur M, Bhattacharjee SK (1988) Herbicide mediated UV-Resistance in cyanobacteria: On the role of photosynthetic electron transport system rather than replicative DNA as lethal target determining dark-survival of Anacystis nidulans. Photochem Photobiol 47:107–113
Domain F, Houot L, Chauvat F, Cassier-Chauvat C (2004) Function and regulation of the cyanobacterial geneslexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation. Mol Microbiol 53:65–80
Dutreix M, Moreau PL, Bailone A, Galibert F, Battista JR, Walker G, Cevoret R (1989) New recA mutations that dissociate the various RecA protein activities in Escherichia coli provides evidence for an additional role for RecA protein in UV mutagenesis. J Bactetriol 171:2415–2433
Duwat P, Eherlich SD, Gruss A (1995) The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress. Mol Microbiol 17:1121–1131
Ganesh N, Muniyappa K (2003) Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA. Biochemistry 42:7216–7225
Gopalakrishna K, Bhattacharjee SK (1976) Role of Rec pathways on sensitivity of Escherichia coli to near-ultraviolet and visible light. J Bacteriol 127:1022–1023
Haefner K (1968) Spontaneous lethal sectoring, a further feature of Escherichia coli strains deficient in the function of rec and uvr genes. J Bacteriol 96:652–659
Herdman M, Janvier M, Rippka R, Stanier RY (1979) Genome size of cyanobacteria. J Gen Microbiol 111:73–85
Hitomi K, Okamoto K, Daiyasu H, Miyashita H, Iwai S, Toh H, Ishiura M, Todo T(2000) Cyanobacterial cryptochrome and photolyase: Characterization of two photolyase-like genes of Synechocystis sp. PCC6803. NAR, 28: 2353–2362
Huang L, McCluskey MP, Ni H, Rossa RA (2002) Global gene expression profiles of the cyanobacterium Synechocystis sp. Strain PCC 6803 in response to irradiation with UV-B and white light.J Bacteriol. 184:6847–6858
Isabelle M, Tu C-J, Grossman A (2004) Effect of high light on transcripts of stress associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313. Microbiology 177:1271–1281
Jansen MAK, Gaba V, Greenberg BN (1998) Balancing plants and UVB radiation: balancing damage, repair and acclimation. Trends Plant Sci 3:131–135
Kameni A-PT, Couture-Tosi E, Saint-Girons I, Picardeau M (2002) Inactivation of Spirochete recA gene results in a mutant with low viability and irregular nucleoid morphology. J Bacteriol 184:452–458
Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3:109–136
Krizek DT (2004) Influence of PAR and UV-A in determining plant sensitivity and photomorphogenic responses to UV-B radiation. Photochem Photobiol 79:307–315
Kuzminov A (1995) Collapse and repair of replication fork in Escherichia coli. Mol Microbiol 16:373–384
Lusetti SL, Cox MM (2002) The Bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Ann Rev Biochem. 71:71–100
Mahajan SK (1988) Pathways of homologous recombination in Escherichia coli. In: Raju Kucheralapati, Smith GR (eds) Genetic Rcombination. American Society for Microbiology, Washington pp 87–140
Mazon G, Lucena S. Campoy S, Fernandez de Henestrosa AR, Candau P, Barber J (2004) LexA-binding sequences in Gram-positive and cyanobacteria are closely related. Mol Gen Genet 271:40–49
Miranda A, Kuzminov A (2003) Chromosomal suppression and removal in Escherichia coli via linear DNA degradation. Genetics 163:1255–1271
Murphy RC, Bryant DA, Porter RD, de Marsac TN (1987) Molecular cloning and characterization of recA Gene from the cyanobacterium Synechococcus PCC 7002. J Bacteriol 171:2739–2747
Murphy RC, Gasparich GE, Bryant DA, Porter RD (1990) Nucleotide sequence and further characterization of the Synechococcus PCC 7002 recA gene: Complementation of a recA mutation by the Escherichia coli recA gene. J Bacteriol 172: 967–976
Pakrasi HB, Williams JGK, Arntzen CJ (1988) Targeted mutagenesis of the psbE and psbF blocks photosynthetic electron transport: evidence for a functional role of cytochrome b-559 in photosystem II. EMBO J 7:325–332
Parshad R, Sanford KK, Jones JM, Tarone R (1978) Fluorescent light-induced chromosome damage and its prevention in mouse cell culture. Proc Natl Acad Sci USA. 75:1830–1833
Porter RD (1988) DNA transformation. In: Packer L, Glazer AN (eds) Methods Enzymol. 167:703–712
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Schidlowski M (1993) The beginnings of life on earth: evidence from the geological records, In: Greenberg JM (eds) The Chemistry of Life’s Origin. Kluwer, Dordrecht, pp 389–414
Schopf JW, Hayes JM and Walter MR (1983) Evolution of Earths earliest ecosystems:Recent progress and unsolved problems. In: Schoff JW (eds) Earth’s Earliest Biosphere. Princeton University Press, pp 361–384
Sciochetti SA, Blakely GW, Piggot PJ (2001) Growth phase variation in cell and nucleoid morphology in Bacillus subtilis recA mutant. J Bacteriol 183:2963–2968
Sharma G, Prabhu KV, Khanna-Chopra R (2004) Decline in reactive oxygen species at low light intensity can overcome necrosis barrier in hybrid wheat. Curr Sci 87:500–503
Webb RB, Lorenz JR (1972) Toxicity of irradiated medium for repair-deficient strains of Escherichia coli. J Bacteriol 112:649–652
Willetts NS, Clark AJ (1969) Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol 100:231–239
Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. In:Packer L and Glazer AN (eds) Methods Enzymol. 167:766–778
Acknowledgements
We thank Rita Mukhopadhyaya for help in sequencing. This work was supported by Council of Scientific and Industrial Research, Government of India, grant schemes. SKB is thankful to S K Mahajan for useful discussions, and to S. Bharti, K.N. Guruprasad and U. Nundy for their support. We are thankful to Anjana Jajoo and Ashish Awadhiya for their help.
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Minda, R., Ramchandani, J., Joshi, V.P. et al. A homozygous recA mutant of Synechocystis PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark. Mol Genet Genomics 274, 616–624 (2005). https://doi.org/10.1007/s00438-005-0054-z
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DOI: https://doi.org/10.1007/s00438-005-0054-z