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 chromosomes. 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, 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 sitepromiscuous integrases and identified possible new ones.

Computationally-guided technology platform for on-demand production of diversified therapeutic phage cocktails

“…The first step in our approach is to use two complementary computational tools (Islander, TIGER) to identify and precisely map the prophages present in genome sequences of bacteria closely related to the pathogen. We have carried this out for 2168 genomes (12), and plan to extend our analysis to all available genome sequences. Preemptive creation of prophage databases in this manner allows for rapid turn-around in phage therapy cases where time is limited -the physician can use the pathogen's genome sequence, 16S rRNA gene sequence, or MLST sequences to place it on a phylogenetic tree, to identify close relatives bearing large numbers of prophages.…”

“…Prophage detection. Two IGE discovery algorithms, Islander (11) and TIGER (12), were applied to 2023 Pseudomonas genomes downloaded from Genbank in October of 2017.…”

“…Islander identifies IGEs with attB in a tRNA or tmRNA gene (11) ( Figure 1A, right); TIGER identifies IGEs with no bias toward attB context (12) ( Figure 1A, left). An additional software module identifies prophages among the IGEs (12). Using this approach, we determined the prophage content of the 26 genome-sequenced Pseudomonas aeruginosa strains that were available from the American Type Culture Collection (ATCC), totaling 44 prophages.…”

“…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 ( Figure 1D) (13).…”

“…We have developed two complementary computational tools, Islander (11) and TIGER (12), that identify and precisely map integrative genetic elements (IGEs), including prophages, within bacterial (and archaeal) genome sequences. This software reveals the bacterial host, complete sequence, and precise ends of each prophage.…”