Bioinformatic characteristics, excision/cyclization activities and prevalence of an ICE element, ICESag1535, in Streptococcus agalactiae isolated from fish

A number of identified integrative and conjugative elements (ICEs) were reported with the exponential expansion of sequenced complete streptococcal genomes. However, the ICEs in Streptococci spp. of the aquatic animal origin remains enigmatic. This paper aims to analyze the bioinformatic characteristics of ICEs originated from aquatic animals, verify the horizontal transfer capabilities, investigate the prevalences in bacteria, predicting their microbial hosts distribution for horizontal diffusion, and detect the contributions to pathogenicity of host bacteria. Integrated genomic approaches were employed to systematically characterize the structural-functional attributes and epidemiological distribution of ICEs, complemented by in vitro functional assays to validate their horizontal gene transfer capacity and molecular mechanisms. A complete ICE element was found in strain WC1535 isolated from Oreochromis niloticus, named ICE_Sag1535_mutT (abbreviation as ICESag1535). Bioinformatic characteristics of ICEs were analyzed, such as the conjugation modules, recombinase composition, variable region function, and ICE homology, etc. ICESag1535, belongs to the ICESa2603 family-like ICEs, with a length of about 74.1 kb, encoding 73 genes, and contains 25 core mobile genes of the Conj_Tn5252 superfamily to form the conjugant modules backbone. The conserved backbone region of the ICESag1535 contains 5 foreign genes insertion hotspots (HS), and the variable region (VR) including 7 transposable insertion elements (IS), forming 3 complex transposons. These HS and VR can be divided into 7 major functional regions, which involve in the functions of ICE, such as the regulation of ICE stability, conjugation coupling, material transmembrane transportation, stress regulation, bacteriocin synthesis and export, integrative and conjugative evolution, host adhesion, etc. Besides, the 3'-terminal of ICESag1535 consists of three structurally similar site-specific serine integrases in series (TSPSI). Cluster analysis of TSPSI revealed that S. agalactiae isolated from Oreochromis niloticus, Lithobates catesbeiana and Lates calcarifer had identical TSPSIs locating within ICESa2603-like ICEs in bacteria which distributed in Northern hemisphere aquatic ecosystem. The ICEs homology analysis indicated that the conserved conjugational transfer backbone of the ICESa2603-like ICEs encoded by human, pig and fish origin. Streptococci spp. were all homologous to Conj_Tn5252 conjugant modules and had the ability to spread across species. The prevalence of ICESa2603-like ICEs in S. agalactiae from Oreochromis niloticus in Guangdong province were detected by PCR. Rate of ICEs carrier in bacteria isolated from 2014 to 2016 was 78.8%, and that of strains isolated from 2020 to 2021 was increased to 96.3%, showing an increasing trend. Finally, the self-excision capability from genome, cyclized, self-transfer and inserted into the recipient bacterium was verified through conjugation transfer ICE from strain WC1535 to Sag158 that isolated from human, and the attL, attR, attB and oriT sites were characterized. After the integration of ICE into Sag158, IS30 element was detected between TSPSI and attR site. The ICESag1535, identified in this study, was a complete ICE element has self-excision and cyclization activities, and cross-species conjugal transfer capability. Varity complex transposons reside in the variable region, which leading to formation of multiple horizontal transfer mechanisms of ICE, increasing the prevalence diversity. Due to the recruitment specialty of IS elements, the variable region of ICEs are characterized by open-ended evolution, which confer the host microbe an unbounded increasing profits for pathogenicity and spreading. This research is of great significance for microbial safety, biological evolution and genetic resources research.