Overview of Salmonella Genomic Island 1-Related Elements Among Gamma-Proteobacteria Reveals Their Wide Distribution Among Environmental Species

The aim of this study was to perform an in silico analysis of the available whole-genome sequencing data to detect syntenic genomic islands (GIs) having homology to Salmonella genomic island 1 (SGI1), analyze the genetic variations of their backbone, and determine their relatedness. Eighty-nine non-redundant SGI1-related elements (SGI1-REs) were identified among gamma-proteobacteria. With the inclusion of the thirty-seven backbones characterized to date, seven clusters were identified based on integrase homology: SGI1, PGI1, PGI2, AGI1 clusters, and clusters 5, 6, and 7 composed of GIs mainly harbored by waterborne or marine bacteria, such as Vibrio, Shewanella, Halomonas, Idiomarina, Marinobacter, and Pseudohongiella. The integrase genes and the backbones of SGI1-REs from clusters 6 and 7, and from PGI1, PGI2, and AGI1 clusters differed significantly from those of the SGI1 cluster, suggesting a different ancestor. All backbones consisted of two parts: the part from attL to the origin of transfer (oriT) harbored the DNA recombination, transfer, and mobilization genes, and the part from oriT to attR differed among the clusters. The diversity of SGI1-REs resulted from the recombination events between GIs of the same or other families. The oriT appeared to be a high recombination site. The multi-drug resistant (MDR) region was located upstream of the resolvase gene. However, most SGI1-REs in Vibrio, Shewanella, and marine bacteria did not harbor any MDR region. These strains could constitute a reservoir of SGI1-REs that could be potential ancestors of SGI1-REs encountered in pathogenic bacteria. Furthermore, four SGI1-REs did not harbor a resolvase gene and therefore could not acquire an integron. The presence of mobilization genes and AcaCD binding sites indicated that their conjugative transfer could occur with helper plasmids. The plasticity of SGI1-REs contributes to bacterial adaptation and evolution. We propose a more relevant classification to categorize SGI1-REs into different clusters based on their integrase gene similarity.


Supplementary Figures 2. BLAST alignment of the nucleotide sequences of the backbones of SGI1-REs with the reference GI as request.
The colors of the strains correspond to the clusters of SGI1-REs: red (cluster1: SGI1 cluster), green (cluster 2: PGI1 cluster), blue (cluster 3: PGI2 cluster), purple (cluster 4: AGI1 cluster), brown (cluster 5), black (cluster 6), and grey (cluster 7). The characterized SGI1-REs are underlined. The ORFs of the backbone used as a reference are represented. The following colors are used for ORFs of known function: The green angled arrows indicate AcaCD binding sites, the red arrow indicates oriT and the black arrow indicates the MDR region. Black vertical lines represent attL and attR attachment sites and the chromosomal trmE gene is shown in black. Backbone variations are described for each SGI1-RE.
(2A) Alignment of SGI1-REs of the SGI1 cluster (cluster 1) with the SGI1 backbone.  Vibrio cholerae N2757 (VSGQ01000007): deletion of part of S006, S007-S013 and part of S014; lower nt identity for the S015-S018 region, but better nt identity with P013-P015; insertion of 2 ORFs sharing nt identity with locus A6033_05025 and a region encoding an ImmA/IrrE family metallo-endopeptidase from Aeromonas veronii CB51 (CP015448) between S018 and S019; lower nt identity for the S021-S022 region; the S023-res region shares nt identity with the Sputw3181_4076 locus of Shewanella sp. W3-18-1 (CP000503), and 3 ORFs encoding an XcyI family restriction endonuclease, a site-specific DNA-methyltransferase and a recombinase; absence of MDR region.
(2B) Alignment of SGI1-REs of the PGI1 cluster (cluster 2) with the PGI1-PmESC backbone.  shares nt identity with the A6033_04990 locus of Aeromonas veronii CB51 (CP015448); region between P010 and P015 shares nt identity with the A6033_05010 and A6033_05015 loci of Aeromonas veronii CB51; longer intergenic region between P016 and P017; P022-res region shares nt identity with S023-S024 followed by 3 ORFs encoding a BsuBI/PstI restriction endonuclease, an N-6 DNA methylase and a resolvase; P025 region corresponds to an ORF encoding a DUF4365 domain-containing protein; absence of MDR region.

PGI1-PmESC
Escherichia coli N63148 (NTMS01000010): very low nt identity with PGI1 for traN; low nt identity with PGI1 for traG; traH found upstream of traG; region between P010 and P015 shares nt identity with the A6033_05010 and A6033_05015 loci of Aeromonas veronii CB51 (CP015448); longer intergenic region between P016 and P017; P022-res region shares nt identity with S023-S024 followed by 3 ORFs encoding a BsuBI/PstI restriction endonuclease, an N-6 DNA methylase and a resolvase; P025 region followed by 3 ORFs encoding proteins of unknown function; absence of MDR region.

IMEVmSCCF01
Vibrio mimicus SCCF01 (CP016383): very low nt identity with PGI1 for traN; low nt identity with PGI1 for traG; traH found upstream of traG; region between P010 and P015 shares nt identity with the A6033_05010 and A6033_05015 loci of Aeromonas veronii CB51 (CP015448); longer intergenic region between P016 and P017; P022-res region shares nt identity with S023-S024 followed by 3 ORFs encoding a BsuBI/PstI restriction endonuclease, an N-6 DNA methylase and a resolvase; P025 region corresponds to an ORF encoding a DUF4365 domain-containing protein; absence of MDR region.

AGI1 (AGI1-B) Vibrio cholerae V060002 (AP018677): deletion between A007 and A009
leading to the loss of A008; deletion of part of A025, and A026-res due to insertion of the MDR region.

AGI2 (AGI1-A) Enterobacter hormaechei EclC2185 (MH545561): deletion between A007 and A009
leading to the loss of A008; deletion of part of A025, and A026-res due to insertion of the MDR region.

Halomonas meridiana Eplume2 (AP022869): insertion of an IS1380 element between traN and I006; I023
region corresponds to an ORF encoding an AAA family ATPase; I027 region shares nt identity with S044; absence of MDR region.

SGI1-RE7MaKG14
Marinobacter adhaerens KG14 (JABEVQ010000005/1) (cluster 7): insertion of an IS element of the IS66 family with an ORF encoding a manganese efflux pump in the region between I002 and rep; no significant similarity for the I013-I014 region; I023-I027 region corresponds to 4 ORFs encoding an AAA family ATPase, a DNA mismatch endonuclease Vsr, a DUF4928 family protein and a DNA (cytosine-5-)methyltransferase; absence of res; absence of MDR region.
Pseudohongiella nitratireducens SCS-111 (LWHN01000034/30/29): insertion of an ISL3-like element between I002 and rep; insertion of an ISL3-like element in I006; no significant similarity for the I013-I014 region; I023 region corresponds to an ORF encoding an AAA family ATPase; I027 region shares nt identity with S044; absence of MDR region.
Pseudohongiella nitratireducens CGMCC 1.15425 (BMIY01000006): insertion of an ISL3-like element between I002 and rep; insertion of an ISL3-like element in I006; no significant similarity for the I013-I014 region; I023 region corresponds to an ORF encoding an AAA family ATPase; I027 region shares nt identity with S044; absence of MDR region.

Supplementary Figures 3. BLAST alignment of the first part of the SGI1-RE backbones (attL-oriT segment).
The colors of the strains correspond to the clusters of SGI1-REs: red (cluster1: SGI1 cluster), green (cluster 2: PGI1 cluster), blue (cluster 3: PGI2 cluster), purple (cluster 4: AGI1 cluster), brown (cluster 5). The characterized SGI1-REs are underlined. The ORFs of the query sequence are represented. The following colors are used for ORFs of known function: The green angled arrows indicate AcaCD binding sites, and the red arrow indicates oriT. The black vertical line represents attL or attR attachment sites (3A) Alignment with the alpA-oriT segment of Aeromonas veronii CB51 (CP015448).