Isolation, nucleotide sequencing and genomic  comparison of a novel Irish ICE from wastewater

Integrating conjugative elements (ICEs) are bacterial self-transmissible mobile genetic elements (MGEs) that integrate into the host’s chromosome, replicate with it and transfer via conjugation. ICEs of the prototype SXT/R391 family are distinguished by possessing an integrase gene (type I-IV) which catalyses integration into either the prfC gene or the serine tRNA gene of the Gammaproteobacteria family of bacteria.

SXT/R391 family ICEs from different environments isolated globally were analysed by comparative genomics to identify core and individual element adaptive genes. Adaptive genes include antibiotic resistance genes, heavy metal resistance genes and bacteriophage defence mechanisms but as yet no identifiable pathogenicity determinants have been observed in spite of ICE identification in major pathogens. From this analysis of fully sequenced ICEs, a link between genes acquired by the ICE and the environmental niches where they reside was observed.

To enhance the data on SXT/R391-like ICEs, we characterized two new SXT/R391 elements of this family, ICEpMERPH and ICEPmiIre01. ICEpMERPH isolated in the River Mersey has previously been reported but not genetically characterized. The adaptive functions of the element, identified by nucleotide sequencing and annotation, were studied followed by phenotypic analysis of a novel arsenate determinant not previously recognized. Comparative genomics with ICER391 revealed that this element contained the core ICE architecture with a number of insertions into hotspots within the element. Phylogenetic analysis revealed that the element was related to ICEApl2 with phylogenetic distance to other ICEs. Elevated levels of resistance to arsenate was detected upon assay with E. coli AB1157 ICEpMERPH strains compared to the isogenic AB1157. As SXT/R391 ICE elements have never been isolated in Ireland, we utilized a directed method for their detection using a PCR probe screening technique to identify sites and bacterial hosts harbouring ICE specific amplicons from a local wastewater plant. This involved an initial screening of wastewater to detect ICE amplicons based on the  conserved integrase gene. This was followed by isolation of specific Gammaproteobacteria, specifically Proteus and Vibrio strains (known to be favoured hosts of SXT/R391-like elements) on selective media. Pools of selected Gammaproteobacteria that gave rise to positive amplicons were identified and colonies fractionated until individual ICE containing isolates were clonal. The use of RFLP analysis revealed the presence of a limited number of amplicon types within these pools suggestive that a relatively small number of ICE types might be present in these  wastewater samples. One clone containing an SXT/R391-like amplicon was characterised further. Whole-genome sequencing revealed the host was Proteus mirabilis, based on the 16s rDNA sequence, and that it contained an integrated 81 kb element, termed ICEPmiIre01 (based on ICE nomenclature) in the prfC gene with 75 open annotated reading frames. The “hotspot regions”, where adaptive genes integrate to construct ICE variation, contained resistance modification systems, toxin-antitoxin systems and a novel BREX (bacteriophage exclusion) system, which was characterized further. Unusually the element did not contain selectable markers such as antibiotic or metal resistance determinants making selection for transfer into more characterised genetic backgrounds difficult. Therefore, we utilized a triparental mating system to transposon tag this novel ICE. Tagging using miniTn5 revealed that the element was transmissible to a marked E.coli strain where its phenotype could be further characterised in a well characterised genetic background. This directed isolation of ICE elements suggests that ICEs are present in the environment without apparent selective pressure but may perhaps contain adaptive functions allowing adaptation to particular environments such as wastewater which are known reservoirs for large bacterial phage populations.