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Lytic Bacteriophage Have Diverse Indirect Effects in a Synthetic Cross-Feeding Community 125

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Lytic Bacteriophage Have Diverse Indirect Effects in a Synthetic Cross-Feeding Community 125 The ISME Journal (2020) 14:123–134 https://doi.org/10.1038/s41396-019-0511-z ARTICLE Lytic bacteriophage have diverse indirect effects in a synthetic cross- feeding community 1,2 2,3 2,4 5 1,2,4 Lisa Fazzino ● Jeremy Anisman ● Jeremy M. Chacón ● Richard H. Heineman ● William R. Harcombe Received: 17 July 2019 / Accepted: 15 August 2019 / Published online: 2 October 2019 © The Author(s) 2019. This article is published with open access Abstract Bacteriophage shape the composition and function of microbial communities. Yet it remains difficult to predict the effect of phage on microbial interactions. Specifically, little is known about how phage influence mutualisms in networks of cross- feeding bacteria. We mathematically modeled the impacts of phage in a synthetic microbial community in which Escherichia coli and Salmonella enterica exchange essential metabolites. In this model, independent phage attack of either species was sufficient to temporarily inhibit both members of the mutualism; however, the evolution of phage resistance facilitated yields similar to those observed in the absence of phage. In laboratory experiments, attack of S. enterica with P22vir phage followed these modeling expectations of delayed community growth with little change in the final yield of 1234567890();,: 1234567890();,: bacteria. In contrast, when E. coli was attacked with T7 phage, S. enterica, the nonhost species, reached higher yields compared with no-phage controls. T7 infection increased nonhost yield by releasing consumable cell debris, and by driving evolution of partially resistant E. coli that secreted more carbon. Our results demonstrate that phage can have extensive indirect effects in microbial communities, that the nature of these indirect effects depends on metabolic and evolutionary mechanisms, and that knowing the degree of evolved resistance leads to qualitatively different predictions of bacterial community dynamics in response to phage attack. Introduction every day [2]. Viral infection of bacterial populations not only impacts the composition of bacterial communities, but Bacteriophage significantly influence microbial community also influences emergent community functions such as the structure and function [1]. Phage limit the size of bacterial rate at which nutrients are converted into biomass [3]. As a populations, which can change microbial community com- result, phage critically influence biogeochemical cycling position. For example, phage kill >20% of marine bacteria [4], biotechnology [5], the food industry [6], and human health [7, 8]. Despite the importance of phage in microbial communities, we cannot reliably predict the impact of phage on the composition or function of communities. As Supplementary information The online version of this article (https:// doi.org/10.1038/s41396-019-0511-z) contains supplementary material, we strive to manage microbial communities, we must which is available to authorized users. improve our understanding of responses to phage infection in multi-species systems. * William R. Harcombe Phage alter competitive bacterial communities by chan- [email protected] ging the species abundance. When phage kill dominant 1 Department of Microbiology and Immunology, University of competitors, weaker competitors that are resistant to phage Minnesota, Minneapolis, MN, USA can flourish, changing species ratios, which can change 2 BioTechnology Institute, University of Minnesota, community function [9–12]. Sometimes, species ratios Minneapolis, MN, USA rapidly revert to pre-phage frequencies once a host evolves 3 College of Continuing and Professional Studies, University of phage resistance, but costs of resistance can generate per- Minnesota, Minneapolis, MN, USA sistent changes in competitive community composition 4 Ecology, Evolution, and Behavior, University of Minnesota, following phage addition [9]. However, phage attack of one Minneapolis, MN, USA species often has little impact on total community biomass. 5 Biology Department, Kutztown University, Kutztown, PA, USA In communities of competitors, a reduction in host biomass 124 L. Fazzino et al. is typically compensated for by the growth of nonhost obligate mutualism in lactose minimal medium, as S. competitors [13]. Taken together, in competitive systems, enterica cannot consume lactose and instead relies on phage alter species ratios, but have little impact on total acetate excreted by E. coli during overflow metabolism. microbial biomass. Grown under these conditions, these bacteria are a simple Much less is understood about how phage influence two-species cooperative community. To this community, cooperative networks in microbial communities. Microbial we added either an E. coli-specific(T7)orS. enterica- communities are often organized into cross-feeding webs in specific(P22vir) lytic phage and tracked community which each species relies on metabolites excreted by others responses (Fig. 1a). As a null hypothesis, we predicted that [14, 15]. Networks of metabolic dependencies have been cross-feeding would constrain species ratios and therefore described in marine, terrestrial, and human-associated targeted phage attack would inhibit growth of the entire microbial communities [14]. While phage are likely pre- community. However, we anticipated that phage resistance sent in all of these systems, the impact of phage on the would evolve, making biomass reduction temporary. We composition and function of cross-feeding microbial com- found that both phage delayed community growth, but munities remains under-studied. neither phage reduced final host yields and T7 infection of However, the response of cross-feeding communities to E. coli led to surprising changes in species ratios. abiotic disturbances may inform how cross-feeding com- munities respond to phage infection. In the absence of disturbance, obligate mutualism typically constrains species Results ratios such that communities converge on an equilibrium ratio from any initial ratio [16, 17]. This constraint on final Resource-explicit model suggests phage have little species ratios means that limiting one species should impact on final community composition indirectly inhibit cross-feeding partners, thereby decreasing total community biomass, but maintaining species ratios We used a resource-explicit model to predict how a coop- [18]. For example, antibiotic treatment of a three-species eratively growing bipartite bacterial community responds to cross-feeding community that inhibited the most sensitive lytic phage attack during batch growth. We modeled den- member inhibited growth of all members of the community sities of E. coli (E), S. enterica (S), and phage (T7 or by depriving community partners of cross-fed nutrients P22vir), as well as cross-fed metabolite concentrations [19]. Therefore, our null hypothesis is that phage infection (Fig. 1a and Supplementary Methods). Growth of each on one member of a cooperative network will limit growth bacterial species was a function of maximum growth rate of the entire cross-feeding network but will not change (μx) and Michaelis–Menten saturation parameters (km)of species ratios. essential metabolites. Bacterial death due to phage infection Yet, the null hypothesis that phage infection will alter was modeled as a linear interaction between phage and host, cooperative community biomass but not composition has and modified by an adsorption (i.e., predation) constant (γ). several underlying assumptions that may not hold. First, it Phage attack generated new phage particles at a rate set by assumes that bacteria obtain nutrients directly from the the burst size (β). Phage-resistant hosts (ER or SR) were secretions of bacterial partners. Yet there is a rich body of initiated at a frequency of 0.1% in each bacterial population literature suggesting that phage-mediated cell lysis releases so that resistance alleles increased in frequency during nutrients into the environment. Indeed, this ‘viral shunt’ is phage infection. The cost of resistance, if any, was repre- thought to play a major role in global nutrient cycling [20– sented by a smaller growth rate (μx) of resistant bacteria. 22] and may alter species interactions [23, 24]. Second, the Parameters were informed by the literature and adjusted to hypothesis overlooks possible ecological consequences of match experimental observations in the absence of phage the evolution of phage resistance. For example, it assumes (Supplementary Table 1). that phage resistance does not alter the exchange of cross- When modeled in the absence of phage, the community fed nutrients. If phage resistance causes changes in either converged to 88% E. coli regardless of the starting bacterial nutrient secretion or uptake, it could alter species ratios, ratio, consistent with previous wet-lab observations (Fig. 1c potentially changing community function. and Supplementary Fig. 1) [17]. In the absence of phage, In this study, we sought to determine the effects of phage sensitive and resistant bacterial genotypes increased in attack on cooperative communities by combining resource- density with relative frequencies determined by the cost of explicit mathematical modeling and wet-lab experiments of resistance (Supplementary Fig. 2). a synthetic cross-feeding co-culture of Escherichia coli and The model predicted that the presence of phage increases Salmonella enterica [17, 25]. An E. coli strain auxotrophic the time required for the community to reach carrying for methionine was paired with a S. enterica strain that was capacity, but has little
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