Abstract
While looking for novel insect-associated phages, a unique siphophage, Nocturne116, was isolated from a deceased local moth specimen along with its host, which was identified by 16S rRNA gene sequencing as a strain of Lactococcus lactis. Next-generation sequencing and the subsequent genome annotation elaborated on herein revealed that the genome of Nocturne116 is a 25,554 bp long dsDNA molecule with 10 bp long 3′ cos overhangs and a GC content of 37.99%, comprising 52 predicted open reading frames. The complete nucleotide sequence of phage Nocturne116 genome is dissimilar to any of the already sequenced phages, save for a distant link with Lactococcus phage Q54. Functions for only 15/52 of Nocturne116 gene products could be reliably predicted using contemporary comparative genomics approaches, while 22 of its gene products do not yet have any homologous entries in the public biological sequence repositories. Despite the public availability of nearly 350 elucidated Lactococcus phage complete genomes as of now, Nocturne116 firmly stands out as a sole representative of novel phage genus.
Highlights
Latvian Biomedical Research and Study Centre, Ratsupites 1 k-1, LV-1067 Riga, Latvia; Abstract: While looking for novel insect-associated phages, a unique siphophage, Nocturne116, was isolated from a deceased local moth specimen along with its host, which was identified by 16S rRNA gene sequencing as a strain of Lactococcus lactis
We report the isolation, genome sequencing and assembly, as well as genomic characterization of a highly divergent novel Lactococcus phage Nocturne116, providing an in-depth elaboration on the rationale behind assigning its open reading frame products with a putative function using comparative genomics approaches employing free and publicly available tools/databases
Corresponding amino acid sequences for the translational product of each ORF was used as a query against: (1) NCBI conserved domain database (CDD) using NCBI conserved domain search [22] under default settings; (2) against the non-redundant protein sequence database using BLASTp [23] with an e-value threshold of 1 × 10−3 ; (3) HHpred [24] against the Protein Data Bank (PDB), Pfam, UniProt-SwissProtviral70 and NCBI CD databases under default parameters
Summary
While searching for novel insect-associated bacteriophages, a dead specimen of local moth species (presumably Lymantria dispar) was collected on the living premises and stored at +4 ◦ C overnight. After sedimentation of solid particles on the bench of Eppendorf 5424 centrifuge (Eppendorf, Hamburg, Germany) at 2348× g for 5 min, 50 μL of supernatant was spread on three Petri dishes with LB agar (g/L: Tryptone—10 (Sigma-Aldrich, St. Louis, MO, USA), Yeast Extract—5 (Sigma-Aldrich), NaCl (Sigma-Aldrich)—10, Bacto Agar (Sigma-Aldrich)—. 5 mL of phage indicator cultures (IC) was prepared in LB liquid media from the single colonies by overnight incubation at RT. To screen the crushed insect suspension for phages, the supernatant was further clarified by centrifugation for 30 min at 8228× g on an Eppendorf 5810R centrifuge (Eppendorf) and filtered through a. Phage Nocturne116 was isolated on the lawn of bacterial indicator culture named “LNT” which formed pale semitransparent colonies, and which was identified later to be a strain of Lactococcus lactis by Sanger-based sequencing [11]. Nocturne116 forms semitransparent plaques both at RT and + 30 ◦ C, with larger plaques formed at 30 ◦ C
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