A composite bacteriophage alters colonization by an intestinal commensal bacterium

Abstract

The mammalian intestine is home to a dense community of bacteria and its associated bacteriophage (phage). Virtually nothing is known about how phages impact the establishment and maintenance of resident bacterial communities in the intestine. Here, we examine the phages harbored by Enterococcus faecalis, a commensal of the human intestine. We show that E. faecalis strain V583 produces a composite phage (ΦV1/7) derived from two distinct chromosomally encoded prophage elements. One prophage, prophage 1 (ΦV1), encodes the structural genes necessary for phage particle production. Another prophage, prophage 7 (ΦV7), is required for phage infection of susceptible host bacteria. Production of ΦV1/7 is controlled, in part, by nutrient availability, because ΦV1/7 particle numbers are elevated by free amino acids in culture and during growth in the mouse intestine. ΦV1/7 confers an advantage to E. faecalis V583 during competition with other E. faecalis strains in vitro and in vivo. Thus, we propose that E. faecalis V583 uses phage particles to establish and maintain dominance of its intestinal niche in the presence of closely related competing strains. Our findings indicate that bacteriophages can impact the dynamics of bacterial colonization in the mammalian intestinal ecosystem.

Fig. 1.

Schematic of the ϕV1 and ϕV7 prophage of E. faecalis V583. The locations of the seven putative prophages of E. faecalis V583 are indicated on the circular chromosome of E. faecalis V583. The ϕV1 and ϕV7 elements are magnified to show their gene organization. Arrows indicate predicted ORFs and are drawn to scale. The arrows highlighted in red show the genes EF2948 (ϕV7 DNA primase) and EF0339 (ϕV1 major capsid) that were mutagenized for this study.

Fig. 2.

E. faecalis V583 produces a ϕV1/7 composite bacteriophage. (A) Southern blot of phage DNA isolated from the culture supernatant of WT E. faecalis V583 (WT) or the isogenic mutant strains ΔϕV7 and ΔEF0339 (ΔϕV1 capsid protein). DNA was digested with NdeI (ϕV1 probe) or NsiI (ϕV7 probe). WT V583 genomic DNA (gDNA) is used as a control. (B) Quantification of phage DNA isolated from WT or ϕV1/7-deficient E. faecalis culture supernatant. The absolute copy number of each prophage was measured using qPCR. (C) Imido black staining of ϕV1/7 phage proteins separated by SDS/PAGE. N-terminal sequencing identified a ∼32 kDa protein as the EF0339 ϕV1 capsid protein. (D) Quantitative plaque assay measuring ϕV1/7 particles from E. faecalis V583 or the ΔϕV1, ΔϕV7, and ΔϕV7 primase mutants using E. faecalis CH188 or ATCC 29212 as indicator strains. nd, not detected. (E) Transmission electron micrograph of a ϕV1/7 particle. Arrows indicate the phage capsid and tail. (F) Southern blot of phage DNA isolated from the culture supernatant of the ϕV7 ΔEF2948 primase mutant E. faecalis strain. WT V583 genomic DNA and phage DNA from WT V583 and ΔϕV7 mutant cultures were probed for comparison. The genomic DNA band detected by the ϕV7 probe has a higher molecular weight because of a chromosomal NsiI site that is outside of the ϕV7 genome.

Fig. 3.

Amino acids enhance the production of ϕV1/7. (A) ϕV1/7 production in defined medium containing varying concentrations of d-glucose as the sole carbon source. (B) ϕV1/7 levels determined by plaque assay in the presence of increasing concentrations of casamino acids in BHI. (C) Quantification of ϕV1/7 particles after the addition of 1% casamino acids (+CA), a defined mixture of purified amino acids (+AA), or no addition. (D) Cell density growth curve of E. faecalis grown in BHI or BHI supplemented with CA or AA. ϕV1/7 numbers are represented as pfus per 10⁶ E. faecalis cells. Statistical analysis was performed using a two-tailed Student t test with a Mann–Whitney correction. Error bars ± SEM. *P < 0.05, **P < 0.005. n = 3–5 experiments performed in duplicate.

Fig. 4.

ϕV1/7 is produced during E. faecalis V583 growth in the mouse intestinal tract. (A) The abundance of ϕV1/7 production was measured from E. faecalis cells grown in broth culture or recovered from the small intestines and colons of germ-free and antibiotic-treated C57BL/6 mice. ϕV1/7 levels were normalized to the total bacterial load from each environment. qPCR was used to measure the absolute abundance of ϕV1/7 DNA and any other E. faecalis V583 prophage DNA from the small intestines (B) or colons (C) of germ-free mice. n = 6–8 for A and n = 2 groups of five pooled mouse intestinal samples for B and C.

Fig. 5.

ϕV1/7 enhances E. faecalis V583 colonization in a competitive ecosystem. (A) Competition between WT E. faecalis V583 or the ΔϕV7 strain in mixed coculture with E. faecalis CH188 in BHI broth. V583 was also competed with ϕV1/7-lysogenized CH188, which is resistant to ϕV1/7-mediated lysis (Fig. S6C). (B) Competition between WT E. faecalis V583 or ΔϕV7 and the susceptible strain CH188 from the feces of germ-free mice 24 h after cocolonization. Competition ratio is the ratio of the parental strain (V583 background) to the susceptible or lysogenized strain. Each data point represents one independent culture or individual mouse. Statistical analysis was done by a two-tailed Student t test. Error bars ± SEM *P < 0.05, **P < 0.005.