Introduction

Raoultella spp. are gram-negative, aerobic, non-motile rods belonging to the family Enterobacteriaceae. Raoultella and Klebsiella are closely related genera, and Raoultella spp. were previously classified as members of the genus Klebsiella; however, in 2001, they were reclassified in the genus Raoultella [1]. Raoultella spp. are a rare source of infection in humans, although serious cases associated with sepsis, bacteremia, urinary tract infection, cholecystitis, immunodeficiency, and cirrhosis have been recorded [2,3,4,5]. Clinical isolates of the genus Raoultella can possess resistance to multiple antibiotics, and alternative therapeutics are therefore required [6]. One such alternative approach could be the use of lytic bacteriophages. However, nucleotide sequence data on bacteriophages specific for Raoultella spp. are limited to phage Ro1 [MG250486]. Here, we report the genome sequence and characteristics of the novel phage RP180, which infects Raoultella sp.

Materials and methods

Raoultella sp. strain CEMTC 2544 was obtained from the Collection of Extremophilic Microorganisms and Type Cultures of the ICBFM SB RAS and cultivated in Luria-Bertani (LB) medium and LB agar at 37 °C. Bacteriophage RP180 was isolated from a wastewater sample. Bacteriophage isolation and characterization of biological properties were performed as described previously [7].

RP180 genome library construction and sequencing were performed as described previously [7]. The genome was assembled de novo using SPAdes software of the Unipro UGENE software package [8]. SPAdes software was applied using default settings, and genome assembly resulted in one contig with an average coverage of 300. Putative open reading frames (ORFs) with a 150-bp minimum size were identified using Vector NTI [9] and UGENE software [8]. These ORFs were compared manually to sequences available in the GenBank database, using the BLASTx algorithm. The presence of tRNA genes was detected using tRNAscan-SE [10]. The Virulence Factor Database (VFDB) was used to search for genes encoding toxins [11]. MAFFT software was used to align genome sequences [12]. The CGView server [13] was used for comparative analysis of the genome of RP180 and those of phage K1G [GU196277], phage Jersey [KF148055], and phage FSL-SP031 [KC139518]. The overall identity of the aligned genome sequences was calculated using BioEdit [14]. A phylogenetic analysis of the RP180 genome and 20 related genomes was performed using PhyML3.0 [15].

Results and discussion

Phage RP180 formed clear plaques with a diameter of about 1─1.5 mm on a lawn of the host strain CEMTC 2544 (Supplementary Fig. 1S). Of the 94 Enterobacteria strains tested, RP180 could only infect Raoultella CEMTC 2544 (Supplementary Table 1S). One-step growth experiments revealed a latent period of 15 min with a burst size of ~40 phage particles per infected cell (Supplementary Fig. 2Sa). A multistep bacterial killing curve for phage RP180 showed that the number of live bacteria decreased dramatically in the three hours after infection and increased slowly thereafter (Supplementary Fig. 2Sb). These results indicate that phage RP180 is a virulent phage.

The RP180 genome is a double-stranded linear DNA molecule with a length of 44,851 bp. The GC content of the RP180 genome is 50.3%, while that of the host genome is 55.5%. The genome of RP180 contains 65 putative ORFs, 35 of which encode proteins whose function could be predicted due to their similarity to known proteins, whereas the other 30 were classified as hypothetical proteins. Genes encoding tRNAs, integrases, and toxins were not found (Fig. 1a). The complete genome sequence of Raoultella phage RP180 was deposited in the GenBank database of NCBI with the accession number MK737937.

Electron microscopy revealed that RP180 has an icosahedral head with a diameter of 55-60 nm, which is connected to a non-contractile tail of approximately 120 nm in length, and a baseplate of 20 nm in width with spikes is located at the end of the tail (Fig. 1b). The morphology and size of the phage particle corresponds to those of Siphoviridae family members [16]. The RP180 genome exhibited the highest similarity to the genomes of the K1G-like phages, which belong to the subfamily Guernseyvirinae, family Siphoviridae. The overall nucleotide sequence identity of the RP180 genome to those of phages representing three genera of the subfamily Guernseyvirinae was 61% for Escherichia phage K1G (genus Kagunavirus), 49% for Salmonella phage Jersey (genus Jerseyvirus) [17], and 40.6% for Salmonella phage FSL-SP031 (genus Cornellvirus). In addition, the RP180 genome showed gene synteny typical of members of the subfamily Guernseyvirinae (Fig. 1a). Phylogenetic analysis of the RP180 genome and other similar phage genomes revealed that phage RP180 is a new member of the genus Kagunavirus (Fig. 2).

Fig. 1
figure 1

Raoultella phage RP180 characteristics. a Genome map prepared using the CGView server. For sequence similarity comparisons, TBLASTX was used to compare K1G (red), Jersey (dark green), and FSL-SP031 (dark blue). b Electron microphotograph of a phage particle

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Fig. 2
figure 2

Phylogenetic analysis performed on the genomes of RP180 and 20 related phages. GenBank identifiers for the sequences are indicated. The investigated sequence is indicated by a black circle. Analysis was performed using the maximum-likelihood (ML) method. Bootstrap values are given at nodes

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The RP180 genome was found to encode a CRISPR-like Cas4 nuclease belonging to the family pfam12705/PDDEXK_1. Cas4-like proteins have been shown to interfere with the host type II-C CRISPR-Cas system [18, 19]. Cas4-like nucleases have been found in phage infecting Campylobacter, Mycobacteria, Propionibacterium, Bacillus, Thermus and other bacteria; however, their exact functions are unknown [20].

In conclusion, a novel phage RP180 infecting Raoultella sp., belonging to the genus Kagunavirus, subfamily Guernseyvirinae was described. Almost all members of the subfamily Guernseyvirinae are Escherichia and Salmonella phages, with only a few exceptions: Xanthomonas phage f29-Xaj [KU595434] [21], Enterobacter phage phiEap-2 [KT287080] [22] and Serratia phage η [KC460990] [23]. Phage RP180 is one more phage of the subfamily Guernseyvirinae that does not infect Escherichia spp. or Salmonella spp.