An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae.

Klebsiella pneumoniae is an important and versatile bacterium that can be found in diverse environments and is also a frequent cause of human infections. Limited data exists on the mechanisms of interaction between K. pneumoniae and the human host and of adaptations to other environments. Coupled with the high genetic diversity of this species, these factors highlight the necessity for substantial further K. pneumoniae-focused molecular genetics studies. In this report we describe a simple and efficient experimental protocol for suicide vector-based allelic exchange in K. pneumoniae. The protocol has been validated by mutating multiple loci in four distinct K. pneumoniae strains, including highly capsulated and/or multi-antibiotic resistant clinical isolates. Three key enhancements are reported:(1) Use of pDS132-derived conjugative plasmids carrying improved cloning sites, (2) Performance of sacB counterselection at 25°C as opposed to higher temperatures, and (3) Exploitation of Flp-recombinase-mediated deletion of FRT (Flp recombinase target) flanked resistance cassettes to allow for reiterative manipulations with a single selectable marker. This study also highlights a problem that may be encountered when the aacC1 gentamicin resistance marker is used in K. pneumoniae and suggests alternative markers. The protocol developed in this study will help investigate the plethora of uncharacterized genes present in the K. pneumoniae pan-genome and shed further light upon clinically and industrially important phenotypes observed in this ubiquitous species.

Expansion of the known Klebsiella pneumoniae species gene pool by characterization of novel alien DNA islands integrated into tmRNA gene sites.

Klebsiella pneumoniae is an important bacterial pathogen of man 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. This organism also occurs naturally in other diverse environments, including the soil. 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 the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species.

The pheV phenylalanine tRNA gene Klebsiella pneumoniae clinical isolates is an integration hotspot for possible niche-adaptation genomic islands.

Horizontally acquired genomic islands may allow bacteria to conquer and colonize previously uncharted niches. Four Klebsiella pneumoniae tRNA gene insertion hotspots (arg6, asn34, met56, and pheV) in 101 clinical isolates derived from blood, sputum, wound, bile or urine specimens were screened by long-range PCR for the presence or absence of integrated islands. The pheV phenylalanine tRNA gene was the most frequently occupied site and harbored at least three entirely distinct types of islands: (1) KpGI-1, a 3.7 kb island coding for four proteins, three of which showed high similarity to two hypothetical proteins and a Gcn5-related N-acetyltransferase in Salmonella enterica, (2) KpGI-2, a 6.4 kb island coding for five proteins including a truncated phage-like integrase, two helicase-related proteins, and a homolog of the functionally elusive Fic protein, and (3) KpGI-3, a 12.6 kb island which carried seven fimbriae-related genes, first identified in MGH78578. Consistent with the niche-adaptation hypothesis, KpGI-1-like islands which coded for the putative acetyltransferase were significantly over-represented in sputum isolates as compared to urine (P < 0.001), blood (P < 0.05) or bile (P < 0.05) derived isolates. Despite the unique nature of KpGI-2, likely homologs of orf5_KpGI-2 that coded for Fic were also found at undefined locations in six other clinical isolates, though none possessed the other KpGI-2 genes. We propose that the pheV-associated islands described in this study may contribute to fine tuning and adaptation of K. pneumoniae strains toward preferred infection and/or colonization pathways.