Exploration On Mobilome Of Important Opportunistic Pathogens

Klebsiella pneumoniae is a frequent cause of opportunistic nosocomial respiratory, urinary, bloodstream and wound infections in man. Colonization of various ecological niches in humans and animals is prevalent. K. pneumoniae is also a common environmental organism with established habitats in soil, water and the surface of plants. At an individual level each genome can be divided into core and accessory regions, giving rise at a population level to a K. pneumoniae-gene pool comprising core genes common to nearly all members of the species and a flexible repertoire, individual genes of which are only represented rarely or sporadically within members of the species. The optional gene inventory which potentially allow host strains to exploit new niches through the acquisition of virulence factors, metabolic pathways, antimicrobial resistance mechanisms and/or cell signaling systems is composed of contributions from plasmids, transposons, integrons, prophages and the evolutionarily enigmatic genomic islands (GIs).In this study, The alien DNA integration in eight tRNA/tmRNA gene hotspots were investigated across28diverse ecovars and pathovars representative of selected species K. pneumonia by simple tRIP and Long-Range PCR strategy. We firstly examined the chromosomal context of the tRNA/tmRNA gene loci using our MobilomeFINDER tool. Eight(thr5, arg6, asn33, asn34, phe55, met56, leu82and tmRNA) were identified to be DNA integration hotspots in this species. Twenty-eight K. pneumoniae strains with environmental and clinical origins were then examined by tRIP PCR using protocols optimized to detect bands of up to～4kb to identify the presence or absence of integrated accessory DNA at each of the eight previously defined tRNA/tmRNA integration hotspots.161out of the256loci screened (62.9%) produced distinct tRIP-PCR products demonstrating the presence of empty tRNA sites (n=108) or those potentially harboring short inserts (n=53) as evidenced by tRIP-PCR amplicons～1-3kb larger than expected for an in silico-indentified empty site. By contrast,95sites (37.1%) failed to produce any tRIP PCR products, consistent with possible occupation of these loci by large DNA elements. The polymorphic patterns of tRNA/tmRNA loci occupation observed support predictions of genome diversity in this species and suggest the possibility of specific island repertoires contributing directly to host adaption to particular niches.In an attempt to capture details of the apparent genomic’dark matter’identified, long-range PCR optimized to produce products of～20kb was employed to further examine the95’occupied’tRNA/tmRNA loci in strains that had yielded no tRIP-PCR amplicons. Nineteen sites produced the bright bands of～6-20kb using the same primer pairs that had been used in the original tRIP-PCR screen, confirming the presence of intervening integrated DNA at these sites. The remaining51sites yielded no detectable long-range PCR products suggesting that these harbored even larger islands or that one or both flanking primers failed to anneal as expected. We are currently undertaking chromosome walking and sequencing experiments to further investigate these loci.In this study the tmRNA gene (ssrA) site was found to exhibit the most diversity among32K. pneumonia strains examined. We such sequenced the five obtained long-range PCR products corresponding to the tmRNA sites, measuring15.4kb in CICC20093(designated tmGI_Kp20093, G+C content=46.4%),17.5kb in CICC10011(tmGI_kp10011, G+C content=46.4%),12.7kb in ATCC49790(tmGI_Kp49790, G+C content=48.6%),9.0kb in HS04044(tmGI_Kp44, G+C content=44.3%) and12.0kb in HS04063(tmGI_Kp63, G+C content=50.3%). Resulting insertion fragments flanking by short DRs exhibited the G+C contents significantly different to that of the fully sequenced K. pneumonia strain MGH78578(57.5). These data are strong evidences that the newly discovered islands are of foreign origins. Remarkably,55of75(73.3%) newly identified genes had no significant homology at nucleotide level with sequences in the known gene pool, suggesting that almost every strain analyzed presented a unique set of integrated DNA fragments in tmRNA gene sites.In addition, we have also investigated the alien DNA fragment insertions at the tRNAcc-identified tRNA/tmRNA gene hotspots in28Pseudomonas aeruginosa clinical strains with variable origins. Interestingly, a11kb natural plasmid pHS87b was found to be inserted into the thr61tRNA genes site as an integrated plasmid. Another26kb natural plasimd pHS87a was also identified in Pseudomonas aeruginosa HS87, which encoded a novel toxin-antitoxin system and multiple drug resistance.In conclusion, this study provides an overview of the presently known species-specific gene pool and reports on a relatively low-cost PCR-based strategy to further expand our view of this genomic space. PCR-based interrogation of selected tRNA and tmRNA sites were performed in diverse environmental and clinical strains of K. pneumoniae and Pseudomonas aeruginosa. Given the simplicity and broadly empowering nature of the strategy used, we propose that similar targeted gene discovery approaches are likely to continue to play major supporting roles even in this post-next generation sequencing era.