Additionally, the fim2 locus was amplified using primers PR1224 and PR1222 and was cloned into pJTOOL-7, a pTRC99a derivative, to create pFim2-Ptrc. A fosmid library representative of KR116 ∆fim2K::kan was constructed using the Epicentre Copy Control Fosmid Library Production kit, with some minor modifications. Briefly, 2.5 μg of genomic DNA was sheared to ~40 kb fragments by pipetting through a 200 μl tip. After end repair,

the DNA was ligated into pCC2FOS and packaged into phages using MaxPlax Lambda I-BET151 Packaging Extracts (Epicentre) which were then used to infect E. coli EPI300-T1R. Marker rescue of kanamycin resistant fosmid clones was performed by plating infected EPI300-T1R cells on LB plates supplemented with chloramphenicol and kanamycin. Selected fosmids were subjected to approximately see more 60-fold coverage Roche 454 pyrosequencing (University of Leicester NUCLEUS Genomics Core Facility). Construction of mutant strains K. pneumoniae KR2107, a spontaneous streptomycin-resistant mutant of KR116, was used as the parent strain for all isogenic mutants. It possessed a 24 h growth curve identical to KR116 and agglutinated guinea pig red blood cells in a similar manner. fim2 was exchanged for a kanamycin resistance cassette by lambda Red-mediated recombination. KR2107 was transformed with pKOBEG-Apra,

a temperature-sensitive AZD9291 supplier plasmid encoding the lambda Red recombinase system, and grown at 30°C find more in LB media supplemented with apramycin and 0.2% arabinose. Electrocompetent KR2107/pKOBEG-Apra cells were prepared according to standard methods and electroporated with an SOE-PCR product comprising a kanamycin resistance gene cassette and targeting flanking homologous sequences (Additional file 1: Figure S1). The KR2107∆fim2 mutant was obtained by selecting on LB media plus kanamycin at 37°C. Loss of pKOBEG-Apra was confirmed by reversion to apramycin sensitivity

and a negative PCR with primers EBGNHe and EBGh3. The KR2107∆fim2 mutant was validated by PCR analysis using primer pairs PR1103-Kn2 (2590 bp) and Kn1-PR1104 (3903 bp). The 2095 bp ∆fim::tet fragment was amplified from C3091∆fim::tet∆mrk::kan using primers UpfimB-F and DwfimK-R and electroporated into arabinose-induced KR2107/pKOBEG-Apra to construct the fim mutant [23]. KR2107∆fim∆fim2 was constructed similarly from a KR2107∆fim/pKOBEG-Apra intermediate strain. KR116 ∆fim2K::kan was constructed by conjugative transfer of the suicide construct pJKO-4a to facilitate allelic exchange ([62]; Additional file 1: Figure S1). Transcriptional analysis of fim2 Total RNA was prepared from KR2107 after growing for 16 h in LB liquid medium (37°C, 200 rpm) using the Norgen Total RNA Purification Kit.