| Klebsiella pneumoniae is an important bacterial pathogen of human that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. Pathogenic and drug-resistant genes can be spread by mobile genetic elements which compose the mobile genome, or ?mobilome·, such as conjugal plasmids, IS elements, integrons, transposons and genomic islands (GIs). Especially, chromosome-carrying genomic islands play a significant role in bacterial evolution, pathogenicity and antibiotic resistance. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands.In this study, eight tRNA/tmRNA gene sites were identified as the integration hotspots for foreign DNA by in silico comparative ananlysis of four sequenced K. pneumoniae genomes (MGH 78578, NTUH-K2044, Kp342 and KCTC2242). Using a tRIP-PCR-based strategy the genomic content of the eight hotspots were investigated in 36 diverse environmental and clinical strains of K. pneumoniae. The presence or absence of integrated accessory DNA reveals the genetic diversity of K. pneumoniae strains under different ecological environments. Three tRNA/tmRNA gene site-specific yeast capture vectors were then constructed and one was used to directional clone six 9~36 kb new genomic islands successfully.Furthermore, we sequenced two tmRNA gene site-associated islands, 9.6-kb island tmGI_Kpw49 of K. pneumoniae w49 isolated from rice rhizosphere and 36.6-kb island tmGI_Kp35 of K. pneumoniae HS04035 isolated from the patient wound secretion. Bioinformatic prediction shows tmGI_Kpw49 is related to L-idonate degradation, and may play an important role in the symbiotic process of w49 with plants; whereas, tmGI_Kp35 is a P2-like phage, which have been widely found among various sequenced Enterobacteriaceae genomes. Comparative analysis of sequenced P2-like phages revealed that they consisted of the conserved core structure and accessory specific genes, such as virulence factors.Based on the results of tRIP-PCR, a multidrug-resistant clinical strain K. pneumonia HS11286 we selected to be completely sequenced, which was isolated from human sputum in 2011 at Shanghai. HS11286 consists of seven circular replicons, including one chromosome and six plasmids. The chromosome (5,332,752 bp, 57.5% G+C content) codes for 5,316 putative proteins, 87 tRNAs, 1 tmRNA and 8 copies of 16S-23S-5S rRNAs. 422 HS11286 strain-speci?c genes were identified from in silico ?subtractive hybridizations·of the HS11286 chromosome against four other completely sequenced K. pneumoniae chromosomes by using mGenomeSubtractor with H-value < 0.42. Interestingly, seven prophage regions were detected, of which three regions were intact. In addition, the HS11286 chromosome contains two novel integrative and conjugative elements. Six plasmids occur naturally in the HS11286 strain: pKPHS1 (122,799 bp, 49.5% G+C content), pKPHS2 (111,195 bp, 53.3% G+C content), pKPHS3 (105,974 bp, 52.5% G+C content), pKPHS4 (3,751 bp, 52.2% G+C content), pKPHS5 (3,353 bp, 42.8% G+C content) and pKPHS6 (1,308 bp, 47.9% G+C content). pKPHS1 codes for a CTX-M-14 extended-spectrumβ-lactamase. pKPHS2 carries the blaTEM-1 gene and the carbapenemase gene blaKPC-2,and has a similar backbone with the recently reported K. pneumoniae plasmid pKP048. pKPHS3 possesses ten important resistance determinants, such as tetG, cat, sul1, dfra12 and aph, and is most similar to a Yersinia pestis plasmid pIP1202. Remarkably, the conjugation transfer genes such as tra in pKPHS2 and pKPHS3 may lead to spread of the multidrug resistance among different genus. The three small plasmids pKPHS4, pKPHS5 and pKPHS6 code for the unknown proteins. To our knowledge, the 1.3 kb pKPHS6 is the smallest K. pneumoniae plasmid ever identified.Comparative analyses of the clinical and environmental K. pneumoniae strains have revealed that this species possesses an extremely plastic genome. Mining the K. pneumoniae genomes will be helpful to reveal the key roles of mobile genetic elements in the adaptive evolution and spread of antibiotic resistance. |
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