New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements

Abstract

Integrative genetic elements (IGEs) are mobile multigene DNA units that integrate into and excise from host bacterial genomes. Each IGE usually targets a specific site within a conserved host gene, integrating in a manner that preserves target gene function. However, a small number of bacterial genes are known to be inactivated upon IGE integration and reactivated upon excision, regulating phenotypes of virulence, mutation rate, and terminal differentiation in multicellular bacteria. The list of regulated gene integrity (RGI) cases has been slow-growing because IGEs have been challenging to precisely and comprehensively locate in genomes. We present software (TIGER) that maps IGEs with unprecedented precision and without attB site bias. TIGER uses a comparative genomic, ping-pong BLAST approach, based on the principle that the IGE integration module (i.e. its int-attP region) is cohesive. The resultant IGEs from 2168 genomes, along with integrase phylogenetic analysis and gene inactivation tests, revealed 19 new cases of genes whose integrity is regulated by IGEs (including dut, eccCa1, gntT, hrpB, merA, ompN, prkA, tqsA, traG, yifB, yfaT and ynfE), as well as recovering previously known cases (in sigK, spsM, comK, mlrA and hlb genes). It also recovered known clades of site-promiscuous integrases and identified possible new ones.

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“…Islander was top ranked by both systems for precision (in the information retrieval sense), with values of 0.971 for GI-positive or -negative segments and 1.000 for gold standards. Using the benchmarks and evaluation tools of Bertelli et al, we measured the precision of TIGER as 1.000 in both systems (12). Thus, our two methods have better precision, by two measures, than any of the other 19 tested methods.…”

“…Islander identifies IGEs encoding a tyrosine integrase with attB in a tRNA or tmRNA gene (13) (Fig. 1A, right); TIGER identifies IGEs encoding both tyrosine and serine integrases with no bias toward attB context (12) (Fig. 1A, left).…”

“…We chose to delete the integrase gene (int), which is essential for lysogeny and readily identifiable. Conveniently for deletion, int is typically located at one end of the prophage genome (12). The integrase gene and its surrounding regions, including the attL and attR sites, were deleted by using PCR primers to generate long, partially overlapping amplicons comprising all included prophage sequences, followed by Gibson assembly of the amplicons and transformation into the target host ( Fig.…”

“…The definition of genomic islands (GIs) has been loosened over time to include any chromosomal DNA segment with evidence of horizontal transfer (11). We have introduced the term integrative genetic elements (IGEs) to mean that subset of GIs whose integration into the chromosome can be ascribed to a self-encoded integrase, either from the tyrosine or serine recombinase families (12). The IGE class includes prophages, integrative and conjugative elements (ICEs), and satellites.…”

“…The IGE class includes prophages, integrative and conjugative elements (ICEs), and satellites. We have developed two complementary computational tools, Islander (13) and TIGER (12), that identify and precisely map IGEs within bacterial (and archaeal) genome sequences. This software reveals the bacterial host, complete sequence, and precise ends of each prophage.…”