Additionally, the fim2 locus was amplified using primers PR1224 and PR1222 and was cloned into pJTOOL-7, a pTRC99a derivative, to selleck 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 Angiogenesis inhibitor 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 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 Glycogen branching enzyme plasmid encoding the lambda Red recombinase system, and grown at 30°C INCB28060 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.