Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

et al. (2013) Klebsiella Phage vB_KleM-RaK2 - A Giant Singleton Virus of the Family Myoviridae. PLoS ONE 8(4): e60717. doi:10.1371/journal.pone.0060717 Klebsiella Phage vB_KleM-RaK2 - A Giant Singleton Virus of the Family Myoviridae Eugenijus S imoliu nas 0 Laura Kaliniene 0 Lidija Truncaite 0 Aurelija Zajanc kauskaite 0 Juozas Staniulis 0 Algirdas Kaupinis 0 Marija Ger 0 Mindaugas Valius 0 Rolandas Mes kys 0 Mark J. van Raaij, Centro Nacional de Biotecnologia - CSIC, Spain 0 1 Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University , Vilnius , Lithuania , 2 Laboratory of Plant Viruses, Institute of Botany, Nature Research Centre, Vilnius, Lithuania, 3 Proteomics Centre, Institute of Biochemistry, Vilnius University , Vilnius , Lithuania At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family. - Funding: This work was funded by the Lithuanian Science Foundation (SVE-04/2012). URL: http://www.lmt.lt/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Bacteriophages (phages) represent the largest reservoir of unexplored genetic information in the biosphere. With biochemical or genetic data being available for a few prototypical phages only, there is a bewildering array of different genomes to characterize [1,2,3,4]. Tailed phages, representing the most diversified of all virus groups, constitute the order Caudovirales with three families, characterised by contractile, long and noncontractile, or short tails and named Myoviridae, Siphoviridae and Podoviridae, respectively [4]. The myoviruses generally have larger genomes than bacteriophages representing other phage families [5]. Moreover, there is a handful of myoviruses with sequenced genomes in the 200 to 500 kbp range. These phages are usually referred to as giant phages or jumbo phages [6], and include such myoviruses as: Pseudomonas aeruginosa phages phiPA3 (309 kbp, [7]) and, phiKZ (280, [8]), Ralstonia phage phiRSL1 (231 kbp, [9]), Yersinia enterocolitica phage R1-37 (270 kbp, [10]). The largest myovirus sequenced to date is Bacillus phage G (498 kbp, #JN638751) followed by Cronobacter phage vB_CsaM_ GAP32 (359 kbp, #JN882285), yet the analysis of these sequences has not been published so far. It is worth mentioning that the vast majority of sequenced giant bacteriophages have no close relatives [6]. Therefore, the evolutionary relationships between these phages and other well-studied myoviruses are difficult to determine. Moreover, in the case of the jumbo phages, the genome anotation using conventional methods (such as comparative genomics or other similar approaches that have proven useful in studying smaller phages) is seriously hampered by a large number of orphan genes. Hence, compared to their smaller brethren, the giant bacteriophages have been less thoroughly researched [6]. Myoviruses infect a broad range of bacterial hosts, including bacteria that prevail in aquatic environments (e.g. cyanobacteria) as well as these that are associated with various human diseases (e.g. pathogenic strains of Escherichia coli, Pseudomonas, Shigella, Klebsiella etc.). Klebsiella are ubiquitously present worldwide. In addition to being the primary cause of various respiratory tract infections, they are frequently associated with the infections of the alimentary, urinary and genital tracts [11]. Moreover, due to the widespread use of antibiotics, multidrug-resistant Klebsiella strains emerge rapidly [12,13,14]. In the past few years, several lytic bacteriophages have been reported to be useful in controlling and detection of this bacterial species, yet studies concerning newly isolated Klebsiella phages have almost exclusively focused on biological characteristics or biodistribution and therapeutic efficacy in animal models [15,16,17]. Moreover, of all the tailed phage genome sequences present in the NCBI database to date, only one is of Klebsiella-specific lytic myovirus, bacteriophage KP15 (174 kbp, #GU295964). Therefore, in the case of this particular group of bacterial viruses, only limited genomic and proteomic data are available, and the molecular mechanisms underlying phage-host interaction are rather obscure [18,19,20]. We present here a novel bacteriophage vb_KleM-Rak2 (Rak2), isolated using a multidrug-resistant Klebsiella sp. veterinary isolate KV-3. Unique morphological and physiological characteristics as well as genomic and proteomic features have been described. With a genome size of 345,809 bp, Rak2 is the third largest myovirus Figure 1. Electron micrographs of Klebsiella phage RaK2. (A and A.1) Phage RaK2 particles adsorbed to the surface of Klebsiella sp. KV-3 cells. (B) Purified phage RaK2 particles (1) and one particle of phage T4 (2). (C) RaK2 particle with contracted (C.1) and extended (C.2) tail. (D) Inner tail tube with baseplate and baseplate-associated ramified tail fiber structures. (E) Baseplate with six long tail fibers. doi:10.1371/journal.pone.0060717.g001 and the largest Klebsiella phage sequenced to date. Moreover, limited homology on the DNA and protein levels indicate that RaK2 has no close phage relatives and therefore is a singleton virus of the family Myoviridae. Materials and Methods Phages and Bacterial Strains Bacteriophage RaK2 was originally isolated from sewagepolluted pond in Lithuania using Klebsiella sp. veterinary isolate KV-3 as the host for phage propagation and phage growth experiments [21]. Bacterial strains used in this study for host range determination are listed in Table S1 [22,23,24,25]. MCIC [26] and SCAI [27] selective media were used for isolation of Klebsiella sp. related bacterial strains. For phage experiments, bacteria were cultivated in Luria-Bertani broth (LB) or LB agar. Bacterial growth was monitored turbidimetrically by reading OD600. An OD600 of 0.4 corresponded to 1.46107 KV-3 cells/ml. To identify the isolates, PCR-amplified 16S rRNA gen (...truncated)