Genetically similar temperate phages form coalitions with their shared host that lead to niche-specific fitness effects

Abstract

Temperate phages engage in long-term associations with their hosts that may lead to mutually beneficial interactions, of which the full extent is presently unknown. Here, we describe an environmentally relevant model system with a single host, a species of the Roseobacter clade of marine bacteria, and two genetically similar phages (ɸ-A and ɸ-D). Superinfection of a ɸ-D lysogenized strain (CB-D) with ɸ-A particles resulted in a lytic infection, prophage induction, and conversion of a subset of the host population, leading to isolation of a newly ɸ-A lysogenized strain (CB-A). Phenotypic differences, predicted to result from divergent lysogenic-lytic switch mechanisms, are evident between these lysogens, with CB-A displaying a higher incidence of spontaneous induction. Doubling times of CB-D and CB-A in liquid culture are 75 and 100 min, respectively. As cell cultures enter stationary phase, CB-A viable counts are half of CB-D. Consistent with prior evidence that cell lysis enhances biofilm formation, CB-A produces twice as much biofilm biomass as CB-D. As strains are susceptible to infection by the opposing phage type, co-culture competitions were performed to test fitness effects. When grown planktonically, CB-A outcompeted CB-D three to one. Yet, during biofilm growth, CB-D outcompeted CB-A three to one. These results suggest that genetically similar phages can have divergent influence on the competitiveness of their shared hosts in distinct environmental niches, possibly due to a complex form of phage-mediated allelopathy. These findings have implications for enhanced understanding of the eco-evolutionary dynamics of host-phage interactions that are pervasive in all ecosystems.

Fig. 1. Overview of roseophage-host system.

a Potential outcomes of superinfection of Sulfitobacter sp. strain CB-D with ɸ-A viral particles. b Within the CB-D genome, prophage D is flanked by 15 bp GC-rich direct terminal repeats (attB), within the 3′ end of a host tRNA-Leu gene. ɸ-A harbors a single copy of this sequence (termed attP). c Alignment of ɸ-A and ɸ-D genomes and recruitment of environmental virome reads. Purple plots show nucleotide identity between phage genomes (scale ranging from 0 to 100%). Open rectangles represent individual ORFs; predicted annotations are provided, where possible. Reads from a North Atlantic, size fractioned virome (~35 kb) (PRJNA47483) that mapped to either genome are shown directly beneath each phage ORF map. Reads were simultaneously recruited to both of the ɸ-A and ɸ-D genomes. Yellow bars represent reads that mapped to either ɸ-A and ɸ-D with equal fidelity (n = 562); the distribution of these reads across the two genomes was randomized. Those fragments in black are specific for either ɸ-A or ɸ-D (n = 161). Refer to Fig. S4 for the full suite of reads recruiting to each phage genome.

Fig. 2. Sulfitobacter strains CB-D and CB-A susceptibility tests with ɸ-D and ɸ-A.

a CB-D growth dynamics of cultures superinfected with ɸ-A (open circles), ɸ-D (open triangles), compared with uninfected controls (closed squares). b CB-A growth dynamics of cultures superinfected with ɸ-D (open triangles), ɸ-A (open circles), compared with uninfected controls (closed squares). Phage gene copies c during superinfection of CB-D with ɸ-A as shown in panel A and d during superinfection of CB-A with ɸ-D as shown in panel B, ɸ-A gene copies (closed circles) and ɸ-D gene copies (closed inverted triangles). Phage gene qPCR data represent sum of intracellular and extracellular gene copies. Averages of biological triplicates are reported for all treatments; technical triplicates were run for all qPCR assays. Error bars denote standard deviations and are obscured by the data markers in some instances. Data for this experiment are provided in Tables S4–S7.

Fig. 3. Relative gene expression of CB-D host and phage gene transcripts 3- and 6-h post superinfection with ɸ-A, relative to non-superinfected controls.

a Fold change of host SOS response genes (recA and lexA). b Fold change of ɸ-A genes, peptidase (pepG) and XRE-like transcriptional regulator (xre). c Fold change of prophage (ɸ-D) genes, peptidase (pepG), ssDNA break repair protein (rad52), and DNA replication and repair protein (ssb). Significant differences (Student’s t tests) are denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant). Averages of biological and technical triplicates are reported for all treatments and error bars denote standard deviations. Data for this experiment are provided in Table S8.

Fig. 4. Physiological characteristics of CB-D and CB-A during different modes of growth.

a Growth dynamics of CB-D (light gray) and CB-A (dark gray) in broth cultures and b biofilms at 24 h. Asterisks denote significant differences as determined by Student’s t tests (*p < 0.05). Averages of biological triplicates and technical triplicates are reported for all treatments. Error bars denote standard deviation and are obscured by the data markers in some instances. All data for this experiment are provided in Tables S9 and S10.

Fig. 5. Head-to-head competition in liquid cultures and biofilms.

a Final (24 h) culture densities of broth CB-A monocultures (dark gray), CB-D monocultures (light gray), and co-cultures (black). Horizontal bar graphs depict ratios of CB-D:CB-A and ɸ-A:ɸ-D in broth co-cultures as determined by qPCR. b Final (24 h) crystal violet biofilm assays for CB-A monocultures (dark gray), CB-D monocultures (light gray), and co-cultures (black) grown as biofilms. qPCR was used to quantify total number of gene copies of CB-D (+ɸ-D):CB-A (+ɸ-A) in co-culture, as represented in horizontal bar graph. Significant differences (Student’s t tests) are denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. not significant). Averages of biological triplicates are reported for all treatments and error bars denote standard deviations. Technical triplicates were run for all qPCR assays and eight technical replicates were run for each biofilm assay. Data for this experiment are provided in Tables S11–S13.