Isolation, nucleotide sequencing and genomic comparison of a Novel SXT/R391 ICE mobile genetic element isolated from a municipal wastewater environment

Abstract

Integrative Conjugative Elements (ICE's) of the SXT/R391 family have largely been detected in clinical or environmental isolates of Gammaproteobacteria, particularly Vibrio and Proteus species. As wastewater treatment plants accumulate a large and diverse number of such species, we examined raw water samples taken from a municipal wastewater treatment plant initially using SXT/R391 family integrase gene-specific PCR probes to detect the presence of such elements in a directed approach. A positive amplification occurred over a full year period and a subsequent Restriction Fragment Length Polymorphism (RFLP) analysis revealed a very limited diversity in the treatment plant examined. Samples demonstrating positive amplification were cultured using Vibrio and Proteus selective media and PCR amplification tracking was utilized to monitor SXT/R391-ICE family containing strains. This screening procedure resulted in the isolation and identification of a Proteus mirabilis strain harbouring an ICE. Whole-genome sequencing of this ICE containing strain using Illumina sequencing technology revealed a novel 81 kb element that contained 75 open reading frames on annotation but contained no antibiotic or metal resistance determinants. Comparative genomics revealed the element contained a conserved ICE core with one of the insertions containing a novel bacteriophage defence mechanism. This directed isolation suggests that ICE elements are present in the environment without apparent selective pressure but may contain adaptive functions allowing survival in particular environments such as municipal wastewater which are reservoirs for large bacterial phage populations.

Figure 1

PCR detection of Irish ICE, M: NEB 1 kb Molecular weight marker, 1: ICEPmiIre01, 2: Positive control (AB1157ICER391) 3: Negative control (dH₂O as template). SYBR Safe stained on 1.2% agarose gel using primers IntFor1 and IntRev1.

Figure 2

Restriction Fragment length polymorphism of the Proteus isolate, ULP014 (integrase positive), M:100 bp NEB Molecular weight marker, 1: Uncut DNA, 2: ULP014 (HpaII), 3: ULP014 (ApoII), 4: ULP014 (TauI), 5: ICER391 (HpaII), 6: ICER391 (ApoI), 7: ICER391 (TauI), 8: Control no enzyme “mock digest”.

Figure 3

Molecular map of ICEPmiIre01 showing the location of the genes associated with the 81 kb MGE. Genes coloured purple are associated with excision, integration and control. Genes coloured yellow are associated with conjugative transfer, all other core genes are coloured white. Genes associated with hotspot and variable regions are coloured red (Constructed from MN520463 using Gene Graphics).

Figure 4

Phylogenetic tree from the maximum-likelihood analysis of the core concatenated proteins of 112 SXT/R391 ICEs including the novel isolate ICEPmiIre01 illustrating its relationship with other family members.

Figure 5

Comparative analysis of the BREX systems present in ICEPmiIre01, ICER391 and ICEVpaCan1, located in HS5 of these ICE elements. Genes coloured moss are associated with brxA (predicted RNA-binding domain), genes coloured brown associated with brxB (unknown function), genes coloured light blue are associated with pglX (DNA methylation domain), genes coloured dark green are associated with pglZ (alkaline phosphatase domain) and brxL (lon-like protease domain), genes coloured yellow are associated with a putative DNA repair and genes coloured white of unknown function.

Figure 6

Examples of the genetic organization of HS2 of SXT/R391 ICE elements containing a T-A system, including ICEPmiIre01, ICER997, ICESXT, ICEVchRua1, ICEVflInd1, ICEApl2, ICEPmiJpn1, ICEVchNig1, ICEVchNep1, ICEVchCHN4210, ICEVchCHNAHV1003, ICEPmiChnBCP11, ICEPvuCHN2213, ICEPmiChn15C1 (direct submission) and ICEPmiChn1. Genes coloured orange are associated with ynd and genes coloured blue are associated with ync. Both genes encode a toxin-antitoxin system.