Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18

Abstract

Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens. IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

The First Cbk-Like Phage Infecting Erythrobacter, Representing a Novel Siphoviral Genus

“…TEM analysis revealed that phage vB_EliS-L02 was a siphovirus, the diameter of the long prolate head was 159.4 ± 4.4 nm, and the length of the tail was 240.0 ± 5.1 nm ( Figure 1A ). The morphology of phage vB_EliS-L02 was remarkably distinct from the only reported Erythrobacter phage vB_EliS-R6L (Lu et al, 2017 ), and it is similar to a few Cbk-like phages, namely, Roseobacter phage DSS3P8 (Zhan et al, 2016 ), Phaeobacter phage MD18 (Urtecho et al, 2020 ), Caulobacter phiCbk-like phage (Gill et al, 2012 ), and Sphingobium phage Lacusarx (Nielsen et al, 2017 ).…”

“…Results of phylogenetic trees based on MCP and portal protein showed that Sphingobium phage Lacusarx was the only phage clustered together with phage vB_EliS-L02. And, these two phages were clustered with some Roseobacter phages according to the phylogenetic tree of portal protein, suggesting a relatively close evolutionary distance among them (Zhan et al, 2016 ; Urtecho et al, 2020 ). Moreover, the evolutionary relationships, based on phoH among vB_EliS-R6L, other viruses, and their hosts, revealed that phage vB_EliS-L02 formed a distinctly separate cluster, with the closest relationship with Sphingobium phage Lacusarx.…”

Diversity, evolution and life strategies of CbK‐like phages

“…Overall, owing to putative attP mutations and recombination, the probability of establishing lysogeny for these phages may be greatly reduced or lost. For example, the CbK‐like phage MD18 was shown to inhibit host growth in liquid culture at a concentration below the plaquing detection limit (200 PFU/mL) (Urtecho et al, 2020).…”