Spatial constraint drives negative frequency dependent selection of phage weaponization

Exposure to phages and biofilm formation are common features of diverse bacterial species. Studying phage-host interaction and population dynamics in biofilms with cellular resolution remains a significant challenge, especially when attempting to recapitulate key features of the natural environments that bacteria and phages occupy. Here we study the population dynamics of phage K139 lysogenized and non-lysogenized Vibrio cholerae when growing in biofilms on the surface of chitin particles under flow of sea water, replicating key features of V. cholerae ecology in the marine environment. We find that lysogenized V. cholerae, via spontaneous lytic induction and phage release, initially kill and displace non-lysogenized bacteria on chitin surfaces. After lysogens become common, however, they no longer tend to displace non-lysogenized cells. Using a combination of modeling approaches and phage K139 derivatives with variable infectivity, we show that the ability of lysogens to displace non-lysogens depends on the ability of phages released by spontaneous induction to reach susceptible non-lysogens. We find that phage access to non-lysogenized bacterial hosts declines once lysogens become common, and this occurs due to the spatial constraints inherent to biofilm growth, as well as to superinfection exclusion, which neutralizes phage particles adsorbed to lysogens surface. Thus, once lysogens comprise the majority of the host population, they can be selected against because they incur the cost of spontaneous induction without gaining the benefit of killing non-lysogen cells via phage release. The balance of cost of lytic induction, phage-mediated killing of non-lysogens, and constraints on phage mobility within host bacterial biofilms therefore impose negative frequency dependent selection for lysogenized cells under physiologically realistic growth conditions. Significance StatementBacteria often produce and live within biofilm communities in natural environments, where they also encounter many threats including bacteriophages. Here we show how temperate phages can alter the fitness of their host via lytic induction and phage release within biofilms of V. cholerae on lab-grown marine snow particles. Using a combination of modeling and imaging experiments, we document that the spatial constraints of biofilm architecture and phage-neutralizing superinfection immunity place limits on the extent to which lysogens can competitively displace non-lysogens via phage release.