Effect of Caenorhabditis elegans age and genotype on horizontal gene transfer in intestinal bacteria

Abstract

Horizontal gene transfer (HGT) between bacteria occurs in the intestinal tract of their animal hosts and facilitates both virulence and antibiotic resistance. A model in which both the pathogen and the host are genetically tractable facilitates developing insight into mechanistic processes enabling or restricting the transfer of antibiotic resistance genes. Here we develop an in vivo experimental system to study HGT in bacteria using Caenorhabditis elegans as a model host. Using a thermosensitive conjugative system, we provide evidence that conjugation between two Escherichia coli strains can take place in the intestinal lumen of N2 wild-type worms at a rate of 10(-3) and 10(-2) per donor. We also show that C. elegans age and genotype are important determinants of the frequency of conjugation. Whereas ∼1 transconjugant for every 100 donor cells could be recovered from the intestine of N2 C. elegans, for the age-1 and tol-1 mutants, the detected rate of transconjugation (10(-3) and 10(-4) per donor cell, respectively) was significantly lower. This work demonstrates that increased recombination among lumenal microbial populations is a phenotype associated with host aging, and the model provides a framework to study the dynamics of bacterial horizontal gene transfer within the intestinal environment.

Figure 1.

Bacterial conjugation can occur in the intestine of older C. elegans. A) C. elegans embryos were grown on NGM agar plates seeded with donor E. coli DY330 strain. At various days of adulthood [e.g., d 4 (L4+4)], worms were washed and transferred to plates seeded with lawns of recipient E. coli OP50 strain. After 24, 48, or 72 h, worms were washed and homogenized by grinding, and the lysates were plated on selective agar to quantify bacterial populations with 3 patterns of antibiotic resistance. B) Worms were initially grown on lawns of donor E. coli DY330 (solid bars) and then moved at different points in worm maturation (L4 stage +2, +3, +4, or +7 d) to plates with lawns of E. coli OP50 (open bars; recipient strain). Graph shows intestinal densities of donor E. coli DY330, recipient E. coli OP50, and transconjugant E. coli (shaded bars) recovered from N2 C. elegans 24 h after shifting the worms to lawns of recipient strain. Data represent means ± sd. C) Worms were grown on donor DY330 (solid circles) and moved on d 4 (L4 stage+4) to lawns of recipient OP50 (open squares). Intestinal densities of the donor E. coli DY330, recipient E. coli OP50, and transconjugant E. coli (shaded triangles) isolated from N2 C. elegans after following the same worm population over the 3 d following transfer are shown. Data represent means ± sd.

Figure 2.

RAPD profiles of donor, recipient, and putative transconjugant strains. The donor E. coli DY330 (letter D) recipient E. coli OP50 (R) and 4 putative transconjugants (T1–T4) were studied by RAPD. Amplifications were performed using primer 1290 (left panel) or primer 1254 (right panel). Lane at left contains a 1-kb molecular size marker (M); lanes C represent the negative controls. Labels at bottom indicate the antibiotic-resistance phenotype of the strains (S, susceptible; R, resistant).

Figure 3.

Bacterial conjugation in the intestinal lumen of C. elegans mutants. Intestinal densities of donor E. coli DY330 (solid circles), recipient-E. coli OP50 (open circles), and transconjugant E. coli (shaded triangles) isolated from: age-1 (A); daf-16 (B); tol-1 (C); or phm-2 (D) mutants. Worms were grown on E. coli DY330, and then on d 4 shifted to lawns of E. coli OP50.

Figure 4.

Host genotype determines the size of the transconjugant E. coli population and the conjugation rate. A) Top panel: intestinal densities of transconjugant E. coli within N2 and defined C. elegans mutants throughout the conjugation assay are highlighted. Bottom panel: transconjugant populations within the intestine of N2 and mutant C. elegans on d 6 of conjugation assay. *P < 0.005 vs. N2 worms. B) Top panel: densities of the donor E. coli strain DY330 within the C. elegans intestine throughout the conjugation assays. Bottom panel: donor populations within the intestine on d 6 of conjugation assay in N2 and mutant C. elegans worms. C) Top panel: conjugation rates within N2 C. elegans and defined mutants throughout the conjugation assay. Bottom panel: bacterial gene exchange in N2 and mutant C. elegans on d 6 of conjugation assay. *P = 0.01, **P = 0.005 vs. N2 worms.