Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus

Abstract

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Keywords: Sphingomonas phages; TEM imaging; burst size; genomic synteny; intergenomic similarity; latency period; lytic phages; narrow host range; non-prolate Siphovirus; wheat flag leaf.

Figure 1

A VIRIDIC heat map [51] showing the intergenomic similarity of the isolated phages, Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, compared with the five closest relatives of each phage. Relevant pairwise nucleotide comparisons were identified and made using BLASTN [52]. Phages isolated in this study are colored green.

Figure 2

Clinker map showing the genomic synteny of the isolated phages, Vexovirus birtae and Molestusvirus kimi, and their closest relatives as well as the observed gene cluster conservation between these genomes. Genome sizes (bp) are listed below each phage. The DNA polymerase encoding gene of Vexovirus birtae and Molestusvirus kimi is distinct from that of the other Sphingomonas phages (red arrow). The red box highlights the queuosine biosynthesis operon. The green, purple, and yellow lines represent the different gene clusters found in the phages.

Figure 3

Plaque and virion morphology features of phages Longusvirus carli (A) and Molestusvirus kimi (B). (Top Insert) Phage plaque morphology on lawns of the bacterial host, Sphingomonas sp. strain LR55. Bars indicate 10 mm. (Bottom Inserts) Phage virion morphology as presented by transmission electron micrographs. Bars indicate 100 nm (C). Phage head and tail sizes analyzed using TEM analysis software. Presented are the mean and standard deviation of at least 10 measured virions per phage sample.

Figure 4

One-step growth curves of phages Longusvirus carli and Molestusvirus kimi. The concentration of total phage particles (i.e., phage progeny) at different timepoints following infection is shown. Data represent the average of three independent experiments. Vertical black bars indicate the standard deviation (SD) of each data point. Adsorption was initiated at Time = 0 min. The red dashed line represents the time point at which the adsorption phase ends (Time = 40 min).