Escherichia coli EC93 deploys two plasmid-encoded class I contact-dependent growth inhibition systems for antagonistic bacterial interactions

Abstract

The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAI^EC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI⁺ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.

Keywords: Escherichia coli; competition; contact-dependent growth inhibition; genome; regulation; toxic potency; toxin.

Fig. 1.

The EC93 genome. Schematic representation of the chromosome and two plasmids (pEC904 and pCP127) of EC93 with cdi and rhs genes shown in green, vgrG genes shown in purple and prophages shown in red. The relative size of plasmids and chromosome illustrations are not to scale.

Fig. 2.

EC93 lacking cdi-1 still outcompetes E. coli MG1655. (a) Average c.f.u. ml^-1 of inhibitor (EC93) and target (MG1655) cells at 0 and 16 h following mixing on M9Glu+casAA solid medium (n=3 biological replicates ±sem). (b) Average competitive index calculated from the data in Fig. 2a. Statistical significance was determined using a two-tailed, unpaired t-test (*P<0.05).

Fig. 3.

EC93 expresses two functional CDI systems on M9Glu+casAA solid medium. (a) Average CIs of inhibitor (EC93) and target (EC93 cdi-1 and cdi-2 mutants) cells after 16 h of co-culture on M9Glu+casAA solid medium (n=3 biological replicates). Target cells were transformed with pCDF empty vector or pCDF vector expressing either CdiI1 or CdiI2. (b) Same co-culturing experiment as in Fig. 3a but with MG1655 as target and EC93 or EC93 cdi mutants as inhibitors (n=3 biological replicates ±sem). Statistical significance was determined by two-tailed, unpaired t-test (*P<0.05).

Fig. 4.

The CdiA-CT1 and CdiA-CT2 toxins are membrane ionophores that require AcrB for activity. Membrane depolarization analyses using DiBAC₄(3) (see Materials and Methods). E. coli MG1655 inhibitor cells expressing the CdiA-CT1, CdiA-CT2 or CdiA-CT^NC101 toxins (see Materials and Methods), depicted at the top of the panels, were mixed with E. coli MG1655 target cells in M9Glu+casAA liquid medium, then analysed by flow cytometry. In the lower three panels, targets expressing cognate CdiI immunity proteins were analysed. (b) Quantification of the data in (a) as the fraction of target cells that were depolarized (n=6 biological replicates ±sem). (c) AcrB-dependence of membrane depolarization. Analysis was carried out as in (a) using E. coli delivering the EC93 CdiA-CT1 or CdiA-CT2 toxins (top of panels) and wild-type or ΔacrB target cells (left of panels). (d) Quantification of the data in (c) as the fraction of target cells that were depolarized (n=4 biological replicates ±sem). Statistical significance was determined by two-tailed, unpaired t-test (*P<0.05, **P<0.005, ***P<0.0005).

Fig. 5.

The cdi1 and cdi2 systems of EC93 are differentially expressed. (a) Schematic representation of the transcriptional reporter constructs used to measure cdi transcriptional activity. (b) Relative transcriptional activity of the cdi-1 and cdi-2 loci in EC93 determined by sYFP2 fluorescence (see Materials and Methods). (c) Quantification of the data presented in (b) (n=3 biological replicates ±sem). (d) Schematic representation of the immunofluorescence (IF) approach used to measure CdiA abundance on the surface of EC93. (e) Relative CdiA surface abundance on EC93 cells (see Materials and Methods). (f) Quantification of the data presented in (e) (n=3 biological replicates ±sem). Statistical significance was determined by two-tailed, unpaired t-test (*P<0.05, **P<0.005, ***P<0.0005).

Fig. 6.

CdiA expression levels correlate with growth inhibition phenotypes for both CdiA-CT1 and CdiA-CT2 toxins. (a) Schematic representation of the two chromosomally integrated cdi loci and the two expression plasmids used to deliver the CdiA-CT1 or CdiA-CT2 toxins to target cells. (b) Expression of CdiA (≈319 kDa) in E. coli MG1655 (cdi ^-), MG1655 with chromosomally integrated cdi-1 or a cdiA1-CT2 chimaera (lacA-cdiBA1-CT1/2), and MG1655 expressing cdi-1 or a cdiA1-CT2 chimaera from a medium-copy plasmid (pcdiBA1-CT1/2) were analysed by SDS-PAGE. Total protein was visualized by SYPRO Ruby staining and relative protein abundance was calculated by densitometry using ImageJ (n=4 biological replicates ±sem). (c) Average CIs of the inhibitor cells, with either chromosomal or plasmid-borne cdiBA1-CT1/2 loci, shown at the top with E. coli MG1655 target cells with cognate BamA^MG1655, or non-cognate BamA^Sty CdiA receptor (bottom key). Inhibitor cells and target cells were co-cultured for 16 h in liquid M9Glu+casAA media (n=4 biological replicates ±sem). Statistical significance was determined by two-tailed, unpaired t-test (*P<0.05, **P<0.005, ***P<0.0005.

Fig. 7.

CdiA-CT2 is a more potent toxin than CdiA-CT1 at any given inhibitor-to-target cell ratio. (a) E. coli MG1655 inhibitor cells containing expression plasmids used to deliver either the CdiA-CT1 or CdiA-CT2 toxins, indicated in the lower box, were mixed with E. coli MG1655 target cells and co-cultured for 16 h in liquid M9Glu+casAA media. CIs (y-axis) were determined at different inhibitor to target ratios (x-axis). Each symbol represents one biological replicate.