Temperate phages limit their own propagation under spatial constraint in biofilms on chitin

Biofilm growth and phage exposure are common to diverse bacterial species. Studying phage–host interaction and population dynamics in biofilms with cellular resolution remains a significant challenge, especially when attempting to recapitulate the natural environments that microbes occupy. Here, we study the population dynamics of phage K139 lysogenized and nonlysogenized Vibrio cholerae when growing in biofilms on the surface of chitin particles in seawater, replicating key features of V. cholerae ecology in the marine environment. We find that lysogenized V. cholerae , via spontaneous lytic induction and phage release, kill and displace nonlysogenized bacteria on chitin surfaces. After lysogens become common, however, they can no longer displace remaining nonlysogenized cells. Using a combination of modeling approaches and microscopy experiments, we show that the lysogens’ capacity to displace nonlysogens depends on the ability of phages released by spontaneous induction to reach susceptible nonlysogens. Phage access to nonlysogenized bacterial hosts declines once lysogens become common, and this occurs due to the spatial constraints inherent to biofilm growth as well as to superinfection immunity, which neutralizes phage particles adsorbed to lysogens. Once lysogens comprise the majority of the host population, they can be selected against, because they still incur the cost of spontaneous induction without gaining the benefit of killing nonlysogen cells via phage release. The cost of lytic induction, phage-mediated killing of nonlysogens, and constraints on phage mobility within host bacterial biofilms together yield population dynamics that are consistent with negative frequency-dependent selection for lysogenized cells under physiologically realistic growth conditions.