The defense island repertoire of the Escherichia coli pan-genome

Abstract

It has become clear in recent years that anti-phage defense systems cluster non-randomly within bacterial genomes in so-called "defense islands". Despite serving as a valuable tool for the discovery of novel defense systems, the nature and distribution of defense islands themselves remain poorly understood. In this study, we comprehensively mapped the defense system repertoire of >1,300 strains of Escherichia coli, the most widely studied organism for phage-bacteria interactions. We found that defense systems are usually carried on mobile genetic elements including prophages, integrative conjugative elements and transposons, which preferentially integrate at several dozens of dedicated hotspots in the E. coli genome. Each mobile genetic element type has a preferred integration position but can carry a diverse variety of defensive cargo. On average, an E. coli genome has 4.7 hotspots occupied by defense system-containing mobile elements, with some strains possessing up to eight defensively occupied hotspots. Defense systems frequently co-localize with other systems on the same mobile genetic element, in agreement with the observed defense island phenomenon. Our data show that the overwhelming majority of the E. coli pan-immune system is carried on mobile genetic elements, explaining why the immune repertoire varies substantially between different strains of the same species.

Fig 1. Schematic of the defense island search approach employed in this study.

(A) Regions containing defense systems in 1,351 E. coli genomes were mapped to the E. coli K-12 genome based on flanking core genes, identifying hotspots for integration of defense-carrying mobile elements. (B) Each hotspot was then searched for in all other E. coli genomes in order to characterize the hotspot occupancy in the E. coli pan-genome. The accession number of each genomic scaffold in the IMG database [23] is shown. Gray shading indicates conservation of core genes flanking the integration position of mobile islands. Known defense system genes are marked in yellow. GInt, Genomic Island with three Integrases.

Fig 2. Occupancy of defense hotspots.

(A) Bar graph showing the occupancy of each integration hotspot among 1,351 E. coli genomes analyzed. The number above each bar indicates the number of genomes in which the hotspot was found occupied. “Hotspot not found” (gray) indicates that one or both core flanking genes were not found in the relevant genome. (B) Nature of mobile genetic elements (MGEs) integrated at hotspots identified in this study. Multiple, analysis of genes in the integrated element suggests a combination of multiple types of MGE.

Fig 3. Examples of mobile islands carrying defense systems with unclear mechanisms of mobilization.

These islands typically contain integrase or recombinase genes but lack other known mobility genes. (A) Selected examples of integrase-only mobile elements integrated at hotspot #2. (B) Selected examples of integrase-only mobile elements integrated at hotspot #33. This hotspot is occupied in E. coli K-12. (C) Selected examples of hotspot #23, comprising defense systems associated with multiple integrases. (D) Selected examples of hotspot #39 occupied by GInt, a newly described Tn6571-family transposon [–36]. Gray shading indicates conservation of core genes flanking the integration position. RM, restriction-modification; hypo., hypothetical gene; Gabija, Hachiman and Zorya are defense systems described in [6]; AVAST was described in [4]; CBASS was described in [13]; Olokun, Menshen, and PsyrTA were described in [7]. Gene symbols of flanking core genes are indicated for each hotspot.

Fig 4. Defense system occupancy at 41 hotspots in the E. coli pan-genome.

Numbers indicate the occurrences of each defense system within each hotspot, with red shading corresponding to the frequency.

Fig 5. Diversity of defense systems at E. coli hotspots.

(A) Number of different defense systems found at each of the 41 hotspots mapped in this study. (B) Defense system distribution for a selected set of the most diverse hotspots.

Fig 6. Hotspot #37 contains an extraordinary diversity of defense systems.

This hotspot is occupied in E. coli K-12.