Cultivation of a Lytic Double-Stranded RNA Bacteriophage Infecting Microvirgula aerodenitrificans Reveals a Mutualistic Parasitic Lifestyle

Bacteriophages are considered the most abundant entities on earth. However, there are merely seven sequenced double-stranded RNA (dsRNA) phages, compared to thousands of sequenced double-stranded DNA (dsDNA) phages. Interestingly, dsRNA viruses are quite common in fungi and usually have a lifestyle of commensalism or mutualism. Thus, the classical protocol of using double layer agar plates to characterize phage plaques might be significantly biased in the isolation of dsRNA phages beyond strictly lytic lifestyles. Thus, we applied a protocol for isolating fungal viruses to identify RNA phages in bacteria and successfully isolated a novel dsRNA phage, phiNY, from Microvirgula aerodenitrificans. phiNY has a genome consisting of three dsRNA segments, and its genome sequence has no nucleotide sequence similarity with any other phage. Although phiNY encodes a lytic protein of glycoside hydrolase, and phage particles are consistently released during bacterial growth, phiNY replication did not block bacterial growth, nor did it form any plaques on agar plates. More strikingly, the phiNY-infected strain grew faster than the phiNY-negative strain, indicating a mutualistic parasitic lifestyle. Thus, this study not only reveals a new mutualistic parasitic dsRNA phage but also implies that other virus isolation methods would be valuable to identify phages with nonlytic lifestyles. IMPORTANCE Viruses with dsRNA genomes are quite diverse and infect organisms in all three domains of life. Although dsRNA viruses that infect humans, plants, and fungi are quite common, dsRNA viruses that infect bacteria, known as bacteriophages, are quite understudied, and only seven dsRNA phages have been sequenced so far. One possible explanation for the rare isolation of dsRNA phages might be the protocol of the double-layer agar plate assay. Phages without strictly lytic lifestyles might not form plaques. Thus, we applied the protocol of isolating fungal viruses to identify RNA phages inside bacteria and successfully isolated a novel dsRNA phage, phiNY, with a mutualistic parasitic lifestyle. This study implies that dsRNA phages without strictly lytic lifestyles might be common in nature and deserve more investigations.