Researchers investigated mechanisms of phage resistance in the marine bacterium Cellulophaga baltica and assessed how they affect the bacterium's role in capturing and exporting carbon to the ocean interior. They found that some resistance mutations do not reduce, and may enhance, the ability of these cells to sink carbon to the seafloor by making the cells more adhesive.
The team identified two main classes of mutations: surface changes that block phage entry altogether and intracellular metabolic mutations that still allow phage entry but prevent successful viral replication. These internal resistance mechanisms are less documented and point to a route where phages can infect the cell but cannot complete their replication cycle.
"We found that both metabolic and surface mutations caused the bacteria to get stickier, but only in surface mutants did those changes cause the cells to sink much more readily. That was very, very obvious," said Marion Urvoy, co-first author of the study and a postdoctoral research associate in microbiology at The Ohio State University. "And that's kind of cool when you think about it, because carbon export in the ocean is important. From past papers, we know that virus abundance is the best predictor of carbon export, more so than any other organism, but we don't know all the mechanisms behind this. It's possible that the selection of surface mutants through infection, which promotes the sinking of bacteria, is one explanation."
The research was published recently in Nature Microbiology. The study focused on 13 phage-resistant mutants of Cellulophaga baltica that evolved against two types of phages representing ecologically relevant model systems.
After infecting the bacteria with these phages and isolating the phage-resistant mutated cells, researchers ran experiments and built computational models to see how the mutants behaved. Results showed that surface mutations that blocked phage entry produced complete resistance to several phages, while internal metabolic mutations provided specific resistance to only one phage at a time.
The team isolated the effect of one of the intracellular changes they observed. This mutation altered the production of a single amino acid that helps synthesize several cellular lipids, molecules that store energy, form membranes and transmit signals.
"The mutation impacted the pool of lipids, which prevented phage replication," Urvoy said. "Our working theory is that because the phage needs these lipids to assemble new virus particles, it is not able to assemble at the end of the replication cycle because one of the key parts is missing."
When the team examined ecological consequences, they found that all resistance mutations carried costs for the bacteria and potentially for the surrounding microbial community. "We showed for all of these mutations, whether they affect the cell surface or its metabolism, there's a cost in terms of growth rates. That is to say the cells are growing slower, and if you affect the growth rate of an organism, you're bound to affect other members of the community," Urvoy said. "We found this decreasing growth was more pronounced for the surface mutants - so they're more resistant to more phages, but it comes at the cost of growing slower in general."
Despite slower growth, surface mutants showed strong increases in stickiness and sinking behavior, which the authors highlight as an important pathway for strengthening the marine biological pump that moves carbon to the deep sea. This builds on earlier work led by co-first author Cristina Howard-Varona, a research scientist in microbiology at Ohio State, indicating that cyanobacteria under simultaneous phage infection and predation stress may increase carbon uptake.
Howard-Varona plans to extend the work on intracellular resistance pathways, which remain sparsely characterized. "This really opens the gate to wanting to examine more intracellular resistance because it's so understudied," she said. "If we add more types of phages, do you get more mutations and more types of mechanisms that we don't know about? This is really just the tip of the iceberg."
Urvoy and Howard-Varona work in the lab of senior study author Matthew Sullivan, professor of microbiology and civil, environmental and geodetic engineering and director of the Center of Microbiome Science at Ohio State. Sullivan's research program investigates how viruses influence microbiomes in ocean, soil and human systems and develops experimental and bioinformatic approaches to track their effects on processes such as carbon cycling.
"It's important to understand what happens in the ocean because it affects climate globally. For microorganisms, we need to understand their impacts on carbon because they dictate whether carbon sinks or gets released into the atmosphere, and that outcome impacts our lives," Urvoy said. "Our work and other work is now showing that viruses, as a component of the marine microbiome, also play roles, perhaps quite centrally, and we need to understand how they affect bacteria and how that fits into the whole picture."
This work was supported by the U.S. National Science Foundation, the U.S. Department of Energy and the Swedish Research Council. Additional co-authors are Carlos Osusu-Ansah, Marie Burris, Natalie Solonenko and Karna Gowda of Ohio State; Andrew Stai and Robert Hettich of Oak Ridge National Laboratory; John Bouranis and Malak Tfaily of the University of Arizona; and Karin Holmfeldt of Linnaeus University in Sweden.
Research Report:Phage resistance mutations in a marine bacterium impact biogeochemically relevant cellular processes
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