Complete Genome Sequence of the Giant Pseudomonas Phage Lu11

Abstract

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 φRSL1, 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.

GENOME ANNOUNCEMENT

The 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, φKZ, 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, Sint-Martens-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 PSI-BLAST (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/extractUpStreamDNA/ [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 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 φRSL1, 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.