Isolation and characterisation of novel phages infectingLactobacillus plantarumand proposal of a new genus, “Silenusvirus”

Abstract

Bacteria of Lactobacillus sp. are very useful to humans. However, the biology and genomic diversity of their (bacterio)phage enemies remains understudied. Knowledge on Lactobacillus phage diversity should broaden to develop efficient phage control strategies. To this end, organic waste samples were screened for phages against two wine-related Lactobacillus plantarum strains. Isolates were shotgun sequenced and compared against the phage database and each other by phylogenetics and comparative genomics. The new isolates had only three distant relatives from the database but displayed a high overall degree of genomic similarity amongst them. The latter allowed for the use of one isolate as a representative to conduct transmission electron microscopy and structural protein sequencing, and to study phage adsorption and growth kinetics. The microscopy and proteomics tests confirmed the observed diversity of the new isolates and supported their classification to the family Siphoviridae and the proposal of the new phage genus "Silenusvirus". Methods Environmental samples, phage assays and bacterial strainsLactobacillus phages were isolated from organic household waste samples. The samples were collected from two different organic waste treatment plants in Denmark (treatment plants A and B). Initially, the samples were split into two subsamples and processed as detailed in Kyrkou et al 12 . The resulting filtrates were screened for phages using the double agar overlay method 16 and a top layer of 0.4% w/v agarose. Specifically, efficiency of plating assays were performed against two indicator strains that had earlier been acquired from private collections, L. plantarum L1 (wine fermentation isolate) and L. plantarum MW-1 (grape isolate). Single plaques were resuspended in 0.7 mL SM buffer and later filtered by 0.45-µm pore size PVDF spin filters (Ciro, Florida, USA). Each purified plaque underwent two further reisolation-filtration cycles to ensure pure stock cultures and was stored at 4°C. For transmission electron microscopy (TEM) and protein sequencing, lysates of 10 10 plaque-forming units (PFUs)/mL) were further purified and concentrated using caesium chloride (CsCl) gradient ultracentrifugation, as described elsewhere 17 . All incubations of phage manipulations were done at 25°C using De Man, Rogosa and Sharpe (MRS) broth and agar media supplemented with 10 mM CaCl 2 (MRSΦ), whereas indicator strains were grown in MRS at 37°C. TEM analysis and structural protein sequencingPhage morphology and structural proteins were determined using the CsCl-purified stocks. Micrographs of phage Silenus were generated as in other studies 12,18 . The mean values and standard deviations of all Silenus virion dimensions were elucidated after inspection of 20-23 phage particles. Structural proteins were sequenced following published protocols 12,18 . Briefly, 100 µL of the CsCl-purified stocks were filtered through an Amicon Ultra filter unit (MWCO 30k Da) and desalted four times. Phage particles (10 µL) were denaturised in 6 M urea, ...