GENOME ANNOUNCEMENT
Genome of Klebsiella sp.-Infecting Bacteriophage vB_KleM_RaK2 Eugenijus Šimoliu nas, Laura Kaliniene, Lidija Truncaite, Vytautas Klausa, Aurelija Zajancˇkauskaite, and Rolandas Meškys VU Institute of Biochemistry, Mokslininku 12, Vilnius, Lithuania
Despite the fact that multidrug-resistant Klebsiella sp. strains emerge rapidly (Xu J, et al., Adv. Mater. Res. 268-270:1954-1956, 2011) and bacteriophages have been reported to be useful in controlling these bacteria (Kumari S, Harjai K, Chhibber S, J. Med. Microbiol. 60:205-210, 2011), the complete genome sequences of only five Klebsiella phages (four siphoviruses and one myovirus) can be found in databases. In this paper, we report on the complete genome sequence of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2. With a genome size of 345,809 bp, this is the second largest myovirus and the largest Klebsiella phage sequenced to date. This phage differs substantially from other myoviruses since 411 out of 534 vB_KleM_RaK2 open reading frames have no known functions and lack any reliable database matches. Comparative analysis of the genome sequence of vB_KleM_RaK2 suggests that this phage forms a distinct phylogenetic branch within the family Myoviridae of tailed bacteriophages.
B
acteriophage vB_KleM_RaK2 (referred to here by its shorter common laboratory name, RaK2) was isolated in Lithuania using Klebsiella sp. veterinary isolate KV-3 as the host for phage propagation. Phage RaK2 is a virulent bacteriophage belonging to the Myoviridae family of viruses. The complete genome sequence of RaK2 was determined using HiSeq 2000 DNA sequencing technology and primer walking. Open reading frames (ORFs) were predicted with Glimmer v2.02 (http: //nbc11.biologie.uni-kl.de). Analysis of the genome sequence was performed using the Fasta-Protein, Fasta-Nucleotide, FastaGenome, BLAST2, PSI-Search, Transeq, and ClustalW2 programs (http://www.ebi.ac.uk), Sequence editor (http://www .fr33.net/seqedit.php) and Geneious v5.5.6. (Société Geneious, Colombes, France). tRNAscan-SE 1.21 (http://lowelab.ucsc .edu/tRNAscan-SE/) was used to search for tRNAs. The circularly permutated linear genome of RaK2 is 345,809 bp long with an overall G⫹C content of 31.7%, which differs significantly from the 57% of Klebsiella spp. With an average ORF size of 583 bp, 91% of the RaK2 genome is coding. It has a total of 534 probable protein-encoding genes, 5 genes for tRNA (tRNRAsn, tRNRArg, tRNRThr, tRNRSer, and tRNRSer2) and 2 pseudo-tRNA genes (tRNRHis and tRNRVal). A total of 531 RaK2 ORFs were found to initiate with AUG, and only 3 were found to initiate with GUG. In contrast to what had been observed in other phages (1, 4), no ORFs of RaK2 were found to initiate from UUG or AUU. Unexpectedly, a marked asymmetry in the distribution of the genes on the two phage DNA strands, with 500 (94%) ORFs on the plus strand and only 34 (6%) ORFs on the minus strand, was observed. The genome analysis revealed that 77% of the RaK2 genes (411 out of 534 ORFs) encode unique proteins that have no reliable identity to database entries. Among the ORFs that encode proteins with matches to those in other sequenced genomes, 99 were similar to proteins from bacteriophages that infect Klebsiella, Pseudomonas, Campylobacter, Synechococcus, Aeromonas, Vibrio, Escherichia coli, and other bacteria. A smaller fraction (24 RaK2 ORFs) was most similar to proteins from Bacteria. Based on the similarity to biologically defined proteins, 68 out of 123 RaK2 ORFs were given a putative functional annotation, including 16 genes coding
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for morphogenesis-related proteins, as well as 19 genes associated with DNA replication, recombination, and repair. Although the gene for the major capsid protein (gp23) of RaK2 is similar to those found in various T4-related bacteriophages, the absence of homologs to essential “core genes” (3, 6, 8, 13, 14, 15, 16, 18, 19, 22, 34, 35, 36, 53, 45, 62, 32, 33, and regA) (5) indicates that this phage cannot be included in the genus of T4-like viruses (3). The analysis of the complete genome sequence revealed that RaK2 is substantially distinct from all of the other characterized myoviruses. Hence, with no close relatives sequenced so far, this phage forms a distinct phylogenetic branch within the family Myoviridae of tailed bacteriophages. Nucleotide sequence accession number. The complete genome sequence of Klebsiella bacteriophage RaK2 was deposited in the EMBL nucleotide sequence database under accession number JQ513383. ACKNOWLEDGMENT This research was funded by a grant (MIP-76/2010) from the Research Council of Lithuania.
REFERENCES 1. Kaliniene L, Klausa V, Zajancˇkauskaite A, Nivinskas R, Truncaite L. 2011. Genome of low-temperature T4-related bacteriophage vB_EcoMVR7. Arch. Virol. 156:1913–1916. 2. Reference deleted. 3. Lavigne R, et al. 2009. Classification of Myoviridae bacteriophages using protein sequence similarity. BMC Microbiol. 9:224. 4. Mann NH, et al. 2005. The genome of S-PM2, a “photosyntethic” T4-type bacteriophage that infects marine Synechococcus strains. J. Bacteriol. 187: 3188 –3200. 5. Petrov VM, Ratnayaka S, Nolan JM, Miller ES, Karam JD. 2010. Genomes of the T4-related bacteriophages as windows on microbial genome evolution. Virol. J. 7:292.
Received 9 February 2012 Accepted 10 February 2012 Address correspondence to Laura Kaliniene,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00347-12
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