Complete Genome Sequence of the Giant Pseudomonas Phage Lu11

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Martens-Latem, Belgium) and ORF Finder (http://www.ncbi.nlm .nih.gov/projects/gorf/ ... BLAST (1), conserved domain search (9), and batch Pfam (10). Con-.
GENOME ANNOUNCEMENT

Complete Genome Sequence of the Giant Pseudomonas Phage Lu11 E. M. Adriaenssens,a,b,c W. Mattheus,a A. Cornelissen,a O. Shaburova,d V. N. Krylov,d A. M. Kropinski,e and R. Lavignea Division of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgiuma; Unit Plant—Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Merelbeke, Belgiumb; Division of Plant Biotechnics, Katholieke Universiteit Leuven, Leuven, Belgiumc; Mechnikov Research Institute for Vaccines & Sera, RAMS, Moscow, Russiad; and Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, and Canada & University of Guelph, Department of Molecular & Cellular Biology, Ontario, Canadae

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ␸RSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.

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he giant bacteriophage Lu11 was isolated in 2002 from a soil sample taken under a mango tree in the Philippines (5). The host of this phage is Pseudomonas putida variant Manila, a common rhizosphere bacterium. We previously described the morphology and proteome of this virus (13). Briefly, Lu11 belongs to the Myoviridae family, with an icosahedral head of 124 nm and a contractile tail 200 nm in length and 20 nm wide. Uniquely structured tail fibers are visible coiling along the tail sheath. Analysis of the structural proteins revealed proteins of similar size as other giant phages, OBP, ␸KZ, and LBG22 (4, 6). Based on its characteristics, Lu11 has been placed as a tentative species in the genus of the “PhiKZ-like viruses” (7). However, the genome data presented here shows that Lu11 is an orphan species within the Myoviridae. The phage was amplified as previously described (13), and DNA was extracted according to reference 11. Sequencing was performed by the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) with 454 technology yielding 41-fold coverage. In parallel, traditional Sanger shotgun sequencing was performed, as described in reference 12, covering ⬎90% of the entire genome. No discrepancies between the 454 and Sanger data were observed. Potential open reading frames (ORFs) were identified using myRAST (2) and then scanned with Kodon (Applied Math, SintMartens-Latem, Belgium) and ORF Finder (http://www.ncbi.nlm .nih.gov/projects/gorf/ [accessed March 2012]) and subsequently curated manually. Putative functions were predicted using PSIBLAST (1), conserved domain search (9), and batch Pfam (10). Conserved promoter sequences were found using a combination of Extract Upstream DNA (http://lfz.corefacility.ca/extractUpStream DNA/ [accessed March 2012]), which extracts the 100-bp upstream sequences of each coding sequence, and MEME/MAST (3) or in Kodon using the early promoter consensus sequence TTGACA (N15-17)TATAAT with a 2-bp mismatch. ARAGORN and tRNAscan-SE were used to search for tRNAs (8, 12). The giant Pseudomonas phage Lu11 has a double-stranded DNA (dsDNA) genome of 280,538 bp with a mol% GC content of 50.8. In total, 291 ORFs were predicted, of which only 44 ORFs had a putative function. Only eight predicted genes coding for parts of the virion particle were found, including tail tube and sheath, baseplate wedge, and tail fibers. The other ORFs with a functional prediction belonged mostly to the nucleotide metabolism and DNA replication genes. Three putative early host-dependent promoters were identified, all located upstream from small hypothetical proteins. A conserved motif of ACTGTAAATA(N7)A, reminiscent of a late T4

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promoter, was discovered upstream from several nucleotide metabolism and DNA replication genes and was labeled as putative middle/late promoter. No tRNAs were discovered. The closest relative of Lu11 is Ralstonia phage ␸RSL1, with 40 ORFs in common based on PSI-BLAST analysis. A CoreGenes analysis (14) was performed and revealed 63 proteins showing similarity. With a similarity less than 20% at the protein level, these phages are only peripherally related, suggesting that Lu11 is a separate species within the Myoviridae. No DNA homology was observed with any phage. Nucleotide sequence accession number. The genome sequence of Pseudomonas phage Lu11 is available under GenBank accession number JQ768459. ACKNOWLEDGMENTS This research was supported by an NSERC Discovery Grant to A.M.K. A.C. holds a predoctoral fellowship from the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWT; Belgium). This research was performed by members of the PhageBiotics research community (reference numbers WO.022.09 and STRT1/10/021TBA).

REFERENCES 1. Altschul SF, Gertz EM, Agarwala R, Schaffer AA, Yu YK. 2009. PSIBLAST pseudocounts and the minimum description length principle. Nucleic Acids Res. 37:815– 824. 2. Aziz RK, et al. 2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75. 3. Bailey TL, et al. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37:W202–W208. 4. Burkal’tseva MV, et al. 2002. Phenogenetic characterization of a group of giant phiKZ-like bacteriophages of Pseudomonas aeruginosa. Russ. J. Genet. 38:1242–1250. 5. Dela Cruz DM. 2004. Master’s thesis. University of Santo Tomás, Espana, Manila, Philippines. 6. Krylov VN, et al. 2004. Comparisons of the genomes of new giant phages isolated from environmental Pseudomonas aeruginosa strains of different regions. Russ.J. Genet. 40:363–368. 7. Krylov VN, Dela Cruz DM, Hertveldt K, Ackermann HW. 2007. “phiKZ-like viruses,” a proposed new genus of myovirus bacteriophages. Arch. Virol. 152:1955–1959.

Received 13 March 2012 Accepted 14 March 2012 Address correspondence to R. Lavigne, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00641-12

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8. Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 32:11–16. 9. Marchler-Bauer A, et al. 2011. CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res. 39:D225–D229. 10. Punta M, et al. 2012. The Pfam protein families database. Nucleic Acids Res. 40:D290 –D301. 11. Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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12. Schattner P, Brooks AN, Lowe TM. 2005. The tRNAscan-SE, snoscan and snoGPS Web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 33:W686 –W689. 13. Shaburova OV, et al. 2006. Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa. Genetika 42:1065–1074. (In Russian.) 14. Zafar N, Mazumder R, Seto D. 2002. CoreGenes: a computational tool for identifying and cataloging “core” genes in a set of small genomes. BMC Bioinformatics 3:12.

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