A Novel and Ubiquitous Marine Methylophage Provides Insights into Viral-Host Coevolution and Possible Host-Range Expansion in Streamlined Marine Heterotrophic Bacteria

Abstract

The methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link between marine primary production and remineralization of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here, we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range. IMPORTANCE Isolation and cultivation of viruses are the foundations on which the mechanistic understanding of virus-host interactions and parameterization of bioinformatic tools for viral ecology are based. This study isolated and characterized the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The nearly identical genomes of four strains of Melnitz isolated from different marine provinces and the global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems, and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning may not be necessarily restricted to phylogenetically related hosts but is a function of shared physical and biochemical properties of the cell.

Keywords: OM43; bacteriophages; cultivation; marine microbiology; virus-host systems.

FIG 1

Viral shared gene content network of OM43 phages, related bacteriophages from the NCBI, and related sequences from the Global Ocean Virome (GOV2.0). Nodes represent viral genomes; edges represent the similarity between phages based on shared gene content. NCBI reference genomes that were greater than more than two neighboring edges from contigs of interest are removed were excluded for clarity. Phage isolates are indicated with red arrows. Colored circles represent genomes and virome contigs within the same cluster as OM43 phage isolate genomes. Nodes shared between Cluster_2 (light pink) and Cluster_3 (purple) are highlighted in hot pink.

FIG 2

Phylogenetic tree of metagenomic contigs and marine Melnitz-type myophages. Neighbor-joining maximum-likelihood tree (500 bootstraps) based on four individually aligned and concatenated structural genes (capsid assembly, major capsid, sheath subtilisin, terminase large subunit) of genomes and contigs that were clustered with OM43 phage Melnitz, with the exception of phages infecting Synechococcus spp. that were used to root the tree. All four Melnitz-like isolates were included but the branch was collapsed for clarity. Branch support values of 1 are not shown. Leaves without labels indicate contigs from the Global Ocean Virome (GOV2) data set (13), which were omitted for clarity. A fully labeled tree is available in Fig. S3 in the supplemental material.

FIG 3

Gene map showing identified genomic features of the OM43 phage Melnitz. (A) Gene map of the 141,548-bp genome of Melnitz contains 143 hypothetical ORFs (62%) without known function (indicated gray). (B) Section of the Melnitz genome between two terminator sequences that contains the ssrA gene, the glutamine riboswitch, and the transcription coactivator gene for transcription-translation regulation.

FIG 4

Structural prediction of CsgGF complex encoded by Melnitz. (A) Predicted structure of CsgGF complex in E. coli (PDB model 6L7A) comprises a hetero-18-mer with 1:1 stoichiometry of CsgG (pink) and CsgF (green), forming a pore in the outer membrane with two constrictions: one provided by CsgG at the base of the barrel and one provided by CsgF at the neck of the barrel. (B) Structural prediction of Melnitz encoded CsgG using AlphaFold2 (teal) showed structural conservation with CsgG from E. coli in the periplasmic α-helices and β-barrel structure. (C) Expanded view of the structural alignment of Melnitz CsgG with E. coli CsgG shows a putative narrowing of the pore at the top of the barrel, matching the pore diameter at the top of the barrel in CsgGF in E. coli. (D) Alignment of predicted structure of Melnitz-encoded CsgF (teal) to that of CsgF in E. coli (pink) showed low structural similarity, with Melnitz-encoded CsgF comprising two alpha-helices and a beta sheet. Alignment indicated that the additional structures of Melnitz-encoded CsgF extend out of the CsgG pore, with unknown function.

FIG 5

Phylogeny of tmRNA genes in major marine lineages. Neighbor-joining maximum-likelihood tree (100 bootstraps) of tmRNA genes found in marine phages and host lineages (not exhaustive) suggests that three the three known major lineages between Cyanobacteria, Gammaproteobacteria, and Alphaproteobacteria are shared with their associated phages, except for OM43 phage Melnitz (infecting H5P1 on the gammaproteobacterial branch), which has a tmRNA gene more closely related to genes found in Alphaproteobacteria and their phages.

FIG 6

Alignment of tRNA genes found in Melnitz, SAR11 and OM43 lineages. Heatmaps and dendrograms (Euclidian similarity matrices) were prepared based on similarity between alignments for arginine (Arg), leucine (Leu), and tryptophan (Trp) tRNA genes found in OM43 phage Melnitz, OM43, and SAR11, as well as tRNA derived from isolated SAR11 and OM43 phages.