Abstract
We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extract. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5′-G/GNCC-3′) in this site to generate a three base 5′ overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 103 viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.
Similar content being viewed by others
References
Biekle TA (1987) DNA restriction and modification. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D.C., pp 692–695
Brown NL, Smith M (1980) A general method for defining restriction enzyme cleavage and recognition sites. Methods Enzymol 65: 391–404
Bryant DA, Tandeau de Marsac N (1988) Isolation of genes encoding components of photosynthetic apparatus. Methods Enzymol 167: 755–765
Buikema WJ, Haselkorn R (1991) Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 173: 1879–1885
Buzby JS, Porter RD, Stevens SE Jr (1983) Plasmid trasformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties. J Bacteriol 154: 1446–1450
Cai Y, Wolk CP (1990) Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol 172: 3138–3145
Currier TC, Wolk CP (1979) Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1. J Bacteriol 139: 82–92
Debus RJ, Barry BA, Babcock GT, McIntosh L (1988) Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 85: 427–430
Blhai J, wolk CP (1988a) A vesatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68: 119–138
Blhai J, Wolk CP (1988b) Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167: 747–754
Blhai J, Wolk CP (1990) Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena. EMBO J 9: 3379–3388
Flores E, Wolk CP (1985) Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. J Bacteriol 162: 1339–1341
Giovanni SJ, Turner S, Olsen GJ, Barns S, Lane DJ, Pace N (1988) Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol 170: 3584–3592
Golden JW, Wiest DR (1988) Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene. Science 242: 1421–1423
Haselkorn R (1978) Heterocysts. Annu Rev Plant Physiol 29: 319–344
Kallas T, Coursin T, Rippka R (1985) Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria. Plant Mol Biol 5: 321–329
Kallas T, Spiller S, Malkin R (1988a) Primary structure of cotranscribed genes encoding the Rieske Fe−S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906. Proc Natl Acad Sci USA 85: 5794–5798
Kallas T, Spiller S, Malkin R (1988b) Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906: highly conserved sequences but different gene organization than in chloroplasts. J Biol Chem 263: 14334–14342
Mannan RM, Whitmarsh J, Nyman P, Pakrasi HB (1991) Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Proc Natl Acad Sci USA 88: 10168–10172
Morrison M, Roderick RI, White BA (1992) Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation. Appl Env Microbiol 58: 66–69
Muro-Pastor AM, Flores E, Herrero A, Wolk CP (1992) Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacteium Anabaena sp. PCC 7120. Mol Microbiol 6: 3021–3030
Murray K, Hughes SG, Brown JS, Bruce SA (1976) Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis. Biochem J 159: 317–322
Oka A, Sugisaki H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147: 217–226
Porter RD (1988) DNA transformation. Methods Enzymol 167: 703–713
Reaston J, Duyvesteyn MGC, deWaard A (1982) Nostoc PCC 7524, a cyanobacterium which contains five sequence-specific deoxyribonucleases. Gene 20: 103–110
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories, and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY
Thiel T, Poo H (1989) Transformation of a filamentous cyanobacterium by electroporation. J Bacteriol 171: 5743–5747
Toelge M, Ziegler K, Maldener I, Lockau W (1991) Directed mutagenesis of the gene psaB of photosystem I of the cyanobacterium Anabaena variabilis ATCC 29413. Biochim Biophys Acta 1060: 233–236
Whitehead PR, Brown NL (1985) Three restriction endonucleases from Anabaena flos-aquae. J Gen Microbiol 131: 951–958
Wolk CP, Vonshak A, Kehoe P, Elhai J (1984) Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci USA 81: 1561–1565
Wolk CP, Cai Y, Cardemil L, Flores E, Hohn B, Murry M, Schmetterer G, Schrautemeier B, Wilson R (1988) Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J Bacteriol 170: 1239–1244
Zhao J, Li N, Warren PV, Golbeck JH, Bryant DA (1992) Sitedirected conversion of a cysteine to aspartate leads to the assembly of a [3 Fe−4 S] cluster in PsaC of photosystem. I. The photoreduction of FA is independent of FB. Biochemistry 31: 5093–5099
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Moser, D.P., Zarka, D. & Kallas, T. Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121. Arch. Microbiol. 160, 229–237 (1993). https://doi.org/10.1007/BF00249129
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00249129