Homing endonucleases encoded in a group I self-splicing intron in a protein-coding gene in cyanophage genomes have not been reported, apart from some free-standing homing edonucleases. In this study, a nicking DNA endonuclease, I-PfoP3I, encoded in a group IA2 intron in the DNA polymerase gene of a T7-like cyanophage Pf-WMP3, which infects the freshwater cyanobacterium Phormidium foveolarum is described. The Pf-WMP3 intron splices efficiently in vivo and self-splices in vitro simultaneously during transcription. I-PfoP3I belongs to the HNH family with an unconventional C-terminal HNH motif. I-PfoP3I nicks the intron-minus Pf-WMP3 DNA polymerase gene more efficiently than the Pf-WMP4 DNA polymerase gene that lacks any intervening sequence in vitro, indicating the variable capacity of I-PfoP3I. I-PfoP3I cleaves 4 nt upstream of the intron insertion site on the coding strand of EXON 1 on both intron-minus Pf-WMP3 and Pf-WMP4 DNA polymerase genes. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a 14 bp region downstream of the cut site. I-PfoP3I requires Mg2+, Ca2+ or Mn2+ for nicking activity. Phylogenetic analysis suggests that the intron and homing endonuclease gene elements might be inserted in Pf-WMP3 genome individually after differentiation from Pf-WMP4. To our knowledge, this is the first report of the presence of a group I self-splicing intron encoding a functional homing endonuclease in a protein-coding gene in a cyanophage genome.
References
[1]
Belfort M, Pedersenlane J, West D, Ehrenman K, Maley G, et al. (1985) Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level. Cell 41: 375–382.
[2]
Landthaler M, Shen BW, Stoddard BL, Shub DA (2006) I-Basl and I-Hmul: Two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities. Journal of Molecular Biology 358: 1137–1151.
[3]
Millard A, Clokie MRJ, Shub DA, Mann NH (2004) Genetic organization of the psbAD region in phages infecting marine Synechococcus strains. Proceedings of the National Academy of Sciences of the United States of America 101: 11007–11012.
[4]
Zeng QL, Bonocora RP, Shub DA (2009) A free-standing homing endonuclease targets an intron insertion site in the psbA gene of cyanophages. Current Biology 19: 218–222.
[5]
Millard AD, Gierga G, Clokie MRJ, Evans DJ, Hess WR, et al. (2010) An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease. Isme Journal 4: 1121–1135.
[6]
Lee CN, Lin JW, Weng SF, Tseng YH (2009) Genomic characterization of the intron-containing T7-like phage phiL7 of Xanthomonas campestris. Applied and Environmental Microbiology 75: 7828–7837.
[7]
Bonocora RP, Shub DA (2004) A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages. Journal of Bacteriology 186: 8153–8155.
[8]
Nord D, Torrents E, Sjoberg BM (2007) A functional homing endonuclease in the Bacillus anthracis nrdE group I intron. Journal of Bacteriology 189: 5293–5301.
[9]
Stoddard BL (2005) Homing endonuclease structure and function. Quarterly Reviews of Biophysics 38: 49–95.
[10]
Colleaux L, Dauriol L, Betermier M, Cottarel G, Jacquier A, et al. (1986) Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into Escherichia-coli as a specific double strand endonuclease. Cell 44: 521–533.
[11]
GoodrichBlair H, Shub DA (1996) Beyond homing: Competition between intron endonucleases confers a selective advantage on flanking genetic markers. Cell 84: 211–221.
[12]
Zhao L, Bonocora RP, Shub DA, Stoddard BL (2007) The restriction fold turns to the dark side: A bacterial homing endonuclease with a PD-(D/E)-XK motif. Embo Journal 26: 2432–2442.
[13]
Biniszkiewicz D, Cesnaviciene E, Shub DA (1994) Self-splicing group I intron in cyanobacterial initiator methionine transfer-RNA - Evidence for lateral transfer of introns in bacteria. Embo Journal 13: 4629–4635.
[14]
Bonocora RP, Shub DA (2001) A novel group I intron-encoded endonuclease specific for the anticodon region of tRNA (fMet) genes. Molecular Microbiology 39: 1299–1306.
[15]
Paques F, Duchateau P (2007) Meganucleases and DNA double-strand break-induced recombination: Perspectives for gene therapy. Current Gene Therapy 7: 49–66.
[16]
Aubert M, Ryu BY, Banks L, Rawlings DJ, Scharenberg AM, et al.. (2011) Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI. Plos One 6.
[17]
Liu XY, Shi M, Kong SL, Gao Y, An CC (2007) Cyanophage Pf-WMP4, a T7-like phage infecting the freshwater cyanobacterium Phormidium foveolarum: Complete genome sequence and DNA translocation. Virology 366: 28–39.
[18]
Liu XY, Kong SL, Shi M, Fu LW, Gao Y, et al. (2008) Genomic analysis of freshwater cyanophage Pf-WMP3 infecting cyanobacterium Phormidium foveolarum: The conserved elements for a phage. Microbial Ecology 56: 671–680.
[19]
Ko M, Choi H, Park C (2002) Group I self-splicing intron in the recA gene of Bacillus anthracis. Journal of Bacteriology 184: 3917–3922.
[20]
Raghavan R, Miller SR, Hicks LD, Minnick MF (2007) The unusual 23S rRNA gene of Coxiella burnetii: Two self-splicing group I introns flank a 34-base-pair exon, and one element lacks the canonical Omega G. Journal of Bacteriology. 189: 6572–6579.
[21]
Johnson RM, Kesavan C, Usha S, Malathi R (2009) Analysis of group I intron splicing in the presence of naturally occurring methylxanthines. Clinica Chimica Acta 400: 74–76.
[22]
Cech TR, Damberger SH, Gutell RR (1994) Representation of the secondary and tertiary structure of group I introns. Nature Structural Biology 1: 273–280.
[23]
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31: 3406–3415.
[24]
Michel F, Westhof E (1990) Modeling of the 3-dimensional architecture of group I catalytic introns based on comparative sequence-analysis. Journal of Molecular Biology 216: 585–610.
[25]
Nikolcheva T, Woodson SA (1997) Association of a group I intron with its splice junction in 50S ribosomes: Implications for intron toxicity. RNA-a Publication of the RNA Society 3: 1016–1027.
[26]
Raghavan R, Hicks LD, Minnick MF (2008) Toxic introns and parasitic intein in Coxiella burnetii: Legacies of a promiscuous past. Journal of Bacteriology 190: 5934–5943.
[27]
Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWweese-Scott C, Geer LY, et al. (2005) CDD: a conserved domain database for protein classification. Nucleic Acids Research 33: D192–D196.
[28]
Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, et al. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Research 31: 1805–1812.
[29]
Mehta P, Katta K, Krishnaswamy S (2004) HNH family subclassification leads to identification of commonality in the His-Me endonuclease superfamily. Protein Science 13: 295–300.
[30]
Landthaler M, Shub DA (2003) The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille. Nucleic Acids Research 31: 3071–3077.
[31]
Landthaler M, Begley U, Lau NC, Shub DA (2002) Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort. Nucleic Acids Research 30: 1935–1943.
[32]
Edgell DR, Belfort M, Shub DA (2000) Barriers to intron promiscuity in bacteria. Journal of Bacteriology 182: 5281–5289.
[33]
Doublie S, Tabor S, Long AM, Richardson CC, Ellenberger T (1998) Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 angstrom resolution. Nature 391: 251–258.
[34]
Shen BW, Landthaler M, Shub DA, Stoddard BL (2004) DNA binding and cleavage by the HNH homing endonuclease I-Hmul. Journal of Molecular Biology 342: 43–56.
[35]
Landthaler M, Lau NC, Shub DA (2004) Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease. Journal of Bacteriology 186: 4307–4314.
[36]
Haugen P, Bhattacharya D, Palmer JD, Turner S, Lewis LA, et al.. (2007) Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns. Bmc Evolutionary Biology 7.
[37]
Bonocora RP, Shub DA (2009) A likely pathway for formation of mobile group I introns. Current Biology 19: 223–228.
[38]
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111: 1–61.
[39]
Wilson WH, Joint IR, Carr NG, Mann NH (1993) Isolation and molecular characterization of 5 marine cyanophages propagated on Synechococcus sp. strain Wh7803. Applied and Environmental Microbiology 59: 3736–3743.
[40]
Rohwer F, Segall A, Steward G, Seguritan V, Breitbart M, et al. (2000) The complete genomic sequence of the marine phage Roseophage SIO1 shares homology with nonmarine phages. Limnology and Oceanography 45: 408–418.
[41]
Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, et al. (2004) Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proceedings of the National Academy of Sciences of the United States of America 101: 11013–11018.
[42]
Horton RM, Ho SN, Pullen JK, Hunt HD, Cai ZL, et al. (1993) Gene-splicing by overlap extension. Methods in Enzymology 217: 270–279.
[43]
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry 72: 248–254.
