Colicins and T6SS-based competition systems enhance enterotoxigenic E. coli (ETEC) competitiveness

Figures & data

Figure 1. Phylogeny and competition factors of ETEC strains.

Relatedness and identified competition factors of 94 ETEC strains and 11 non-ETEC E. coli and Shigella strains using E. fergusonii as outgroup. All ETEC strains are named after respective assembly RefSeq accession number or TW name. Names of non-ETEC strains are in red. Some branches were collapsed based on the presence of bacteriocins and T6SS and named by a representative member of the group. Number of strains present in a collapsed branch is indicated. Green indicates the presence of a bacteriocin or T6SS. Half-filled box indicates that some but not all members of the group (collapsed branches) carry the bacteriocin or T6SS (Full tree in Fig. S1). Yellow indicates partial T6SS system.

Figure 2. Conservation and selective pressure of type i1 T6SS components in ETEC strains with potentially active systems.

a) Nucleotide sequence complexity for each T6SS component in strains with potentially active systems. b) dS/dN values for the unique sequences for each T6SS component. Dashed line = 1 and dS/dN above 1 suggest stabilizing selection. c) Number of codons in the nucleotide sequence of each component with indication of stabilizing or diversifying selection. Gene order corresponds to the T6SS locus represented by TW10722 type i1 in d.

Figure 3. Representative domain composition of putative T6SS effectors identified in ETEC strains with potentially active T6SS (N = 47).

Putative function, how many strains it was identified in and representative accession is summarized for each type of effector. Rhs effectors are grouped based on their genomic location. Details of each Rhs locus is found in Fig. S5.

Figure 4. ETEC TW10722 uses Colicin Ia to outcompete MG1655 in vitro.

a-b) Competitive index of ETEC strains co-cultured at a ratio of 1:1 with E. coli MG1655 on M9-glycerol solid media for 24 h as such (A) or supplemented with 0.2% L-arabinose or glucose (B). Strains were separated by plating on different antibiotics. Error bars represent SEM of N = 6 biological replicates. a) ETEC co-cultured against E. coli MG1655, b) ETEC TW10722 wild-type or col-Ia:cat mutant co-cultured with MG1655 supplemented with empty vector or vector encoded Colicin Ia immunity under an arabinose inducible promoter. Statistical significance was determined using Student’s t-test for A and two-way Anova with Fisher’s LSD post hoc test for B. ns= not significant, *= P < 0.05, **= P < 0.01, ***= P < 0.001 and ****= P < 0.0001. Significance stars above the bars indicate significant difference compared to the MG1655 vs MG1655 control competition (A) or for that competition in the two different media (M9 Glu and M9 Ara) (B). Other relevant statistical analyses are indicated with lines connecting the two samples that were compared. All statistics can be found in Table S7. c) The growth of E. coli MG1655 in M9-glycerol supplemented with spent media from wildtype E. coli MG1655, TW10722, or TW10722 col-Ia:cat cultures. Growth is measured by OD₆₀₀ measurements over a 6 h period.

Figure 5. ETEC TW10828 uses T6SS to outcompete MG1655 in vitro.

a) Illustrations of the T6SS loci found in the different ETEC strains available for competitions (TW10722, TW10828, TW10573, and TW10590). b) Competitions with ETEC with or without tssM against E. coli MG1655 on solid M9-glycerol media supplemented with 0.1% bile salts and L-arabinose for 6 h. Strains were mixed 10:1 and competitive index was calculated as the change in ratio between 0 h and 6 h. Error bars represent SEM, n = 6. Statistical significance was determined using two-way ANOVA with a Fisher LSD post hoc test. ns= not significant, *= P < 0.05, **= P < 0.01, ***= P < 0.001 and ****= P < 0.0001. Significance stars above the bars indicate significant difference compared to the MG1655 vs MG1655 control competition in that media. Other relevant statistical analyses are indicated with lines connecting the two samples compared to each other. All statistics can be found in Table S7. c) T6SS effectors encoded by the ETEC strains used in competitions in B.

Data availability statement

All raw data for the manuscript are freely available either as supplementary excel files (Tables S5–8) or at the NCBI Reference Sequence Database (GCF_032367395.1, GCF_032365155.1, GCF_032366635.1, GCF_032368225.1).