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Genomic heterogeneity has been shown to be associated with Klebsiella pneumoniae strains causing pyogenic liver abscesses (PLA) and metastatic infections. In order to explore the mechanism responsible for genomic heterogeneity in K. pneumoniae , we compared the complete genomic sequences of strains NTUH-K2044 and MGH78578. An ∼76-kbp DNA fragment located adjacent to an asparagine ( asn ) tRNA gene was present in NTUH-K2044 but not in MGH78578. This fragment could be divided into three regions with different functions, and structurally it resembled a functional integrative and conjugative element (ICE), ICE Ec1 , in Escherichia coli . The 5′ region of this fragment contained genes similar to a high-pathogenicity island (HPI) of Yersinia pestis and Yersinia pseudotuberculosis . The middle region was similar to part of a large plasmid in K. pneumoniae , and the 3′ region contained genes responsible for DNA conjugative transfer. Therefore, this DNA fragment was designated ICE Kp1 . Precise excision and extrachromosomal circularization of ICE Kp1 were detected in K. pneumoniae wild-type strain NTUH-K2044. ICE Kp1 could integrate into the asn tRNA loci of the chromosome of another K. pneumoniae isolate. The prevalence of ICE Kp1 was higher in PLA strains (38 of 42 strains) than in non-tissue-invasive strains (5 of 32 strains). Therefore, ICE Kp1 may contribute to the transmission of the HPI and result in K. pneumoniae PLA infection-associated genomic heterogeneity.

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA Asn but by specific tRNA-dependent transamidation of aspartyl-tRNA Asn . This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA Asp and tRNA Asn with aspartic acid. Recently, it has been shown for the first time in an organism ( Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA Asn but does not participate in Gln-tRNA Gln formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA Asn . Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA Asp charging over that of tRNA Asn by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA Asp and tRNA Asn . Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.