Escherichia coli genes affecting recipient ability in plasmid conjugation: are there any?

Abstract

Background: How does the recipient cell contribute to bacterial conjugation? To answer this question we systematically analyzed the individual contribution of each Escherichia coli gene in matings using plasmid R388 as a conjugative plasmid. We used an automated conjugation assay and two sets of E. coli mutant collections: the Keio collection (3,908 E. coli single-gene deletion mutants) and a collection of 20,000 random mini-Tn10::Km insertion mutants in E. coli strain DH5alpha. The combined use of both collections assured that we screened > 99% of the E. coli non-essential genes in our survey.

Results: Results indicate that no non-essential recipient E. coli genes exist that play an essential role in conjugation. Mutations in the lipopolysaccharide (LPS) synthesis pathway had a modest effect on R388 plasmid transfer (6 - 32% of wild type). The same mutations showed a drastic inhibition effect on F-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative DNA processing or transport per se.

Conclusion: We conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. This is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell.

Figure 1

Analysis of conjugation by the light emitted in the HTS assay. (A) The time-course of light emission in 94 individual DH5α wt conjugation mixtures (blue diamonds), donor alone (UCDPM1; red dash) and DH5α expressing Eex_R388 [DH5α (pSU5024); black circle] is shown. (B) Representation of the results in (A) as arbitrary light units [ALU = log₁₀ (Light-x/Light-wt)]. In Fig. 1A light emission of only 20 of the 94 DH5α colonies are represented in order to help in visualization.

Figure 2

ALU values of Keio collection mutants. ALU values [ALU = log₁₀ (Light-x/Light-wt)] of the HTS conjugation assay when applied to the 3,908 Keio collection mutants (grey diamonds), BW25113 wt strain (black triangle), donor without recipient (UCDPM1; black dash) and BW2511 expressing Eex_R388 [BW25113 (pSU5024); black circle)]

Figure 3

Distribution of mutants ALU values. Distribution of ALU values [ALU = log₁₀ (Light-x/Light-wt)] of the HTS conjugation assay when applied to the 3,908 Keio collection mutants (white bars) or to 20,000 miniTn10::kan insertion mutants (grey bars).

Figure 4

Result of the HTS conjugation assay when applied to a selected subset of the 20,000 mini-Tn10::Km insertion mutants. The HTS conjugation assay was run on a collection of 20,000 mini-Tn10::Km insertion mutants as described in Methods. A subset of 237 mutants, which gave ALU ≤ -1 in the first assay were selected for a second assay. The figure shows the ALU values [ALU = log₁₀ (Light-x/Light-wt)] of these 237 mutants plotted in increased order (grey diamonds), together with donor without recipient (UCDPM1; black dash) and DH5α expressing Eex_R388 [DH5α (pSU5024); black circle].

Figure 5

Insertions located in LPS biosynthesis genes. Genetic localization of 11 mini-Tn10::Km insertions in E. coli genes involved in three LPS biosynthesis operons. Transposon insertion positions are shown by triangles that identify mutant numbers.

Figure 6

ALU values of the F-lux-monitored conjugation assay. ALU values [ALU = log₁₀ (Light-x/Light-wt)] of the F-lux-monitored conjugation assay when applied to the LPS mutants (grey diamonds) in solid (A) or liquid (B) mating conditions. DH5α wt strain (black triangle), donor without recipient (black dash) and DH5α [(pOX38); black circle].