Complete nucleotide sequences and annotations of φ673 and φ674, two newly characterised lytic phages of Corynebacterium glutamicum ATCC 13032

The genomes of two new lytic phages of Corynebacterium glutamicum ATCC 13032, φ673 and φ674, were sequenced and annotated (GenBank: MG324353, MG324354). Electron microscopy studies of both virions revealed that taxonomically they belong to the Siphoviridae family and have a polyhedral head with a width of 50 nm and a non-contractile tail with a length of 250 nm. The genomes of φ673 and φ674 consist of linear double-stranded DNA molecules with lengths of 44,530 bp (G+C = 51.1%) and 43,193 bp (G+C = 50.7%) and identical, protruding, cohesive 3’ ends 13 nt in length. The level of identity between the φ673 and φ674 genomes is 85.2%. Two major structural proteins of each virion were separated via SDS-PAGE and identified using peptide mass fingerprinting. Based on bioinformatic analysis, 56 and 54 ORFs were predicted for φ673 and φ674, respectively. Only 20 of the putative gene products of φ673 and 20 of φ674 could be assigned to known functions. Both genomes were divided into functional modules. Nine putative promoters in the φ673 genome and eight in the φ674 genome were predicted. One bidirectional Rho-independent transcription terminator was identified and experimentally confirmed in each phage genome. Electronic supplementary material The online version of this article (10.1007/s00705-018-3867-x) contains supplementary material, which is available to authorized users.


Introduction
Corynebacterium glutamicum is a nonpathogenic, gram-positive bacterium that is widely used for the industrial production of a broad range of substances, including amino acids and proteins [1]. In many cases, phages are responsible for the lysis of commercially interesting strains during fermentation, which leads to financial losses in the biotechnology industry. Many corynephages have been isolated, but only a few of them have been completely sequenced [2][3][4]. In the present study, the genomes of φ673 and φ674, two newly identified lytic phages of C. glutamicum ATCC 13032, were sequenced and annotated. The phages φ673 and φ674 were obtained from VKPM (the Russian National Collection of Industrial Microorganisms at the Institute of Genetics and Selection of Industrial Microorganisms, Moscow). Four genes associated with sensitivity to φ674 were identified in the C. glutamicum ATCC 13032 genome and could be useful for the construction of phage-resistant strains [5]. The newly constructed cosmid based on cos-sites of φ674 could be helpful for improving genetic tools for C. glutamicum, particularly with respect to the non-specific transduction of DNA fragments between C. glutamicum ATCC 13032 strains; such transduction has been reported for other phagehost systems [6,7].

Results and discussion
Phages φ673 and φ674 were propagated on C. glutamicum ATCC 13032 and purified via centrifugation in a CsCl gradient as previously described [8].
Transmission electron microscopy studies of these two phages revealed that their virions belong to the Siphoviridae family. Both virions had a polyhedral head with a width of 50 nm and a long non-contractile tail with a length of 250 nm and a diameter of 11 nm (Fig. 1a, b). The putative gene products (gp) gp φ673 14 and gp φ674 14 were assigned to the tail tape measure protein (TMP). For both phages, the relationship between the observed tail length (~ 250 nm) and TMP size (1,577 aa for φ673 and 1,572 aa for φ674), which involved a ratio of 0.159 nm/aa, was reasonable [9]. Purified genomic DNA from both phages was sequenced using Illumina technology at Evrogen (Moscow, Russia, http://www.evrog en.ru). Sequences of cos-sites were determined in run-off experiments and were compared with the nucleotide sequences of the ligated phage ends.
The φ673 and φ674 genomes consist of linear double-stranded DNA molecules with lengths of 44,530 bp (G+C = 51.1%) and 43,193 bp (G+C = 50.7%), respectively, and share identical, protruding, cohesive 3' ends 13 nt in length (AGA AGG GGG CGG A-3'). A cosmid vector for molecular cloning has been constructed on the basis of the phage φ674 cos-site, and the functionality of the cos-site was experimentally confirmed (unpublished results). Based on bioinformatics analysis, 56 and 54 ORFs were identified in the φ673 and φ674 genomes, respectively. These ORFs cover approximately 97% and 96% of the entire φ673 and φ674 genomes, respectively. Only 20 gene products (gps) from each phage could be assigned to known biological functions (Supplementary Table 1, 2); the other 17 and 16 gp(s) exhibiting homology to hypothetical proteins, while 19 and 18 ORFs present in φ673 and φ674, respectively, had no homologues in the databases. No tRNA genes were identified in either phage genomes.
Nine and eight putative promoters were predicted in the φ673 and φ674 genomes, respectively (Supplementary Table 3, 4). One bidirectional, rho-independent transcription terminator was identified in each phage genome (Supplementary Fig. 2) and experimentally confirmed (unpublished result).
Based on homology to known phage proteins, functional domains, and mutual arrangement, putative functions were assigned to products of 20 of the predicted ORFs in each phage (Supplementary Table 1, 2). For each phage, the entire genome was divided into four functional modules (Fig. 2). The DNA packaging module includes small (gp φ673 1 and gp φ674 1) and large (gp φ673 2 and gp φ674 2) terminase subunits and a portal protein (gp φ673 3 and gp φ674 3). A head maturation protease (gp φ673 4 and gp φ674 4), major capsid and tail proteins (gp φ673 5 and gp φ674 5 and gp φ673 11 and gp φ674 11), (c, f) the corresponding predicted amino acid sequences (not highlighted) and the aa sequences detected in the analysis (highlighted) are shown φ673 and φ674, lytic phages of Corynebacterium glutamicum head-to-tail connectors (gp φ673 7, 8, 9 and gp φ674 7, 8, 9), a tail assembly chaperone (gp φ673 12 and gp φ674 12), a tail TMP (gp φ673 14 and gp φ674 14), a tail protein (gp φ673 16 and gp φ674 16) and a tail fiber protein (gp φ673 19, 21 and gp φ674 19) could be predicted in the structural components and assembly module. Two major structural proteins for each virion, the major capsid (gp φ673 5 and gp φ674 5) and tail (gp φ673 11 and gp φ674 11) proteins, were detected via SDS-PAGE and identified via trypsin-based peptide mass fingerprinting (PMF) using an Ultraflex II LC-MALDI-TOF/TOF (Bruker) in accordance with a previously described procedure [10] (Fig. 1c, d, e). Furthermore, elimination of an N-terminal Met residue retained in trypsin-digested peptides from gp φ673 11 and gp φ674 11 confirmed the predicted N-terminal processing rule [11] (Fig. 1c, e).
A homolog of a known enzyme, lysozyme-like protein (gp φ673 22 and gp φ674 20), was predicted in the host lysis module. The replication/recombination/metabolism module also contained homologs to known proteins, including helicase (gp φ673 43 and gp φ674 40), the DNA replication protein RepA primase/helicase (gp φ673 45 and gp φ674 42), DNA polymerase I (gp φ673 46 and gp φ674 44) and HNH endonuclease (gp φ673 33 and gp φ674 30, 31, 43). One transcriptional regulator, gp φ673 27, was identified. Interestingly, a putative intein was identified in the helicase encoded in ORF 43 for φ673, in contrast to the helicase encoded in ORF 40 for φ674, which exhibited no inteins. It has previously been reported that the Corynebacterium phage P1201 contains inteins [3].
Significant similarity throughout the genome was observed between the two newly sequenced and annotated lytic corynephages, φ673 and φ674, which exhibited approximately 85.2% identity. A bioinformatics search revealed that both phage genomes had high similarity to the genome of the corynephage BFK20 [2], with approximately 55% identity. Multiple genome alignment was constructed with Mauve (ver. 2.2.0) (Supplementary Fig. 3).
Besides C. glutamicum ATCC 13032, the host strain for both φ673 and φ674 phages, MB001 (prophage-free variant of C. glu ATCC 13032) was also infected by both phages. Another tested wild-type strain Brevibacterium lactofermentum AJ1511 was not lysed by either of the two phages.
We identified four C. glutamicum ATCC13032 genes, responsible for phage φ674 sensitivity (unpublished results). Two of these genes encoded glycosyltransferases; these proteins are bacterial sugar transferases involved in lipopolysaccharide synthesis. The third gene is annotated as a gene encoding a putative secreted protein. The fourth gene encodes a nucleotidyltransferase/DNA polymerase involved in DNA repair that is a DNA polymerase IV homolog. We hypothesized that these glycosyltransferases participate in the synthesis of a φ674 phage receptor containing an unknown sugar component in its structure [12].
In summary, the genomes of the φ673 and φ674 phages are significantly different from existing corynephage genomes available in databases; therefore, the sequences of these complete phage genomes were deposited for the first time in Gen-Bank under accession numbers: MG324353, MG324354.