Prophage contribution to bacterial population dynamics

Abstract

Cocultures of Salmonella strains carrying or lacking specific prophages undergo swift composition changes as a result of phage-mediated killing of sensitive bacteria and lysogenic conversion of survivors. Thus, spontaneous prophage induction in a few lysogenic cells enhances the competitive fitness of the lysogen population as a whole, setting a selection regime that forces maintenance and spread of viral DNA. This is likely to account for the profusion of prophage sequences in bacterial genomes and may contribute to the evolutionary success of certain phylogenetic lineages.

FIG. 1.

Evolution of the ratio of strains in cocultures of S. enterica serovar Typhimurium strains carrying or lacking the Gifsy-2 prophage. Strains were derived from S. enterica serovar Typhimurium strain ATCC 14028s (3). Cultures (in Luria-Bertani medium) were inoculated with approximately 1,000 CFU from a 1:10 mixture of bacteria lysogenic for Gifsy-2 or lacking the prophage, respectively. Inoculi were verified to be phage-free. After the bacteria reached stationary phase, cultures were diluted 200-fold and incubated further under the same conditions (30°C, no shaking) for 6 to 12 h. The subculturing routine was repeated the indicated number of times (number [N.] of generations). At each dilution step, the culture was analyzed with respect to bacterial composition and phage concentration. (A) The lysogenic strain carries wild-type Gifsy-2. (B) The lysogenic strain contains a Gifsy-2 prophage variant that cannot excise due to an int-xis deletion. Lysogenization by the indicated phage is indicated by a double colon. The full genotypes for competing strains follow: MA5973, lacking Gifsy-1 but carrying Gifsy-2 (Gifsy-2[+]); MA6055, lacking Gifsy-1 and Gifsy-2 but with ara-907 araD901::MudJ; MA7434, lacking Gifsy-1 but with Δ[int-xis]^Gifsy-2::kan. (The int-xis/kan swap construct was obtained as described previously [1]. The deleted segment corresponds to the interval between coordinates 1098338 and 1099776 of Salmonella strain LT2 genome sequence [8].) Phage indicator strain MA6684 lacked Gifsy-1 and Gifsy-2 but had galE496 bio-106::Tn10. Panels A and B show the results of one representative experiment of three and two independent experiments, respectively.

FIG. 2.

Evolution of the ratio of strains in cocultures of S. enterica serovar Typhimurium strains carrying or lacking the Gifsy-1 and Gifsy-2 prophages. The experiment was performed as described in the legend to Fig. 1. (A) The naive strain, MA6055, is susceptible to infection by both Gifsy-1 and Gifsy-2 phages. (B) The naive strain, MA7467, carries an ompC gene disruption that renders the strain resistant to Gifsy-1 and Gifsy-2 infection. Lysogenization by the indicated phage(s) is indicated by a double colon. The full genotypes of competing strains follow: MA5958, carrying Gifsy-1 and Gifsy-2 (Gifsy-1[+]Gifsy-2[+]); MA6055, see the legend to Fig. 1; MA7467, lacking Gifsy-1 and Gifsy-2 but with ompC159::Tn10 ara907 araD901::MudJ. The full genotypes of phage indicator strains follow: MA6685, lacking Gifsy-1 but carrying Gifsy-2 galE496 bio-106::Tn10; MA7416, carrying Gifsy-1 and lacking Gifsy-2 but carrying galE496 bio-106::Tn10. Panel A shows the result of one representative experiment of two independent experiments.

FIG. 3.

Plaques from phage with Gifsy-2 immunity in early and late subcultures. Supernatants from cocultures of strains MA5958 and MA6055 were diluted suitably and plated on a lawn of strain MA7416 (Fig. 2). (A) Phage from dilution step 3 in Fig. 2A. (B) Phage from dilution step 6 in Fig. 2A. The larger plaques have the size and morphology typical of Gifsy-1 phage.

FIG. 4.

Evolution of the ratio of strains in cocultures of S. enterica serovar Typhimurium strains from unrelated lineages. Strain MA6711 (a gift from S. Uzzau, University of Sassari, Sassari, Italy) is a phage-type DT104 strain from an epidemic outbreak in Zimbabwe. The experiment was performed as described in the legend to Fig. 1, except that strains were inoculated in a 1:1 ratio. Phage titers were measured by plating aliquots from culture supernatants onto bacterial lawns from strains MA6711 (total phage from MA6054 or MA6055) and MA5958 (total phage from MA6711). (A) Strains harbor the wild-type prophage complements of the respective lineages. (B) The ATCC 14028s-derived strain (MA6055) lacks the Gifsy-1 and Gifsy-2 prophages.