The deletion of gplH in antibiotic sensitive clones was screened for and confirmed by PCR. Towards this end, chromosomal DNA isolated from mutant candidates was used as template along with primer pairs (pepOF and

pepOR, pepF and pepR) that produced diagnostic amplicons permitting differentiation between the mutant and WT genotypes. Construction Ganetespib mw of p2NIL-GOALc-ΔgplHc and pCP0-gplH The plasmid p2NIL-GOALc-ΔgplHc used in the construction of Ms ΔgplH carried the gplH deletion cassette ΔgplHc. The deletion cassette contained: 995-bp segment upstream of gplH + gplH’s first 13 codons (5 fragment) followed by gplH’s last 4 codons + stop codon + 1,000-bp segment downstream of gplH (3 fragment). ΔgplHc was built by the joining of the 5′ fragment and the 3′ fragment using splicing-by-overlap-extension (SOE) PCR [59]. Each fragment was PCR-generated from chromosomal DNA. Primer pair pepOF and pepIR and primer pair pepIF and pepOR were used to generate the 5’ and 3’ fragments, respectively. The fragments were then used as template

for PCR with primers pepOF and pepOR to fuse the fragments and create ΔgplHc (2,061 Palbociclib mouse bp). The PCR-generated ΔgplHc was first cloned into pCR2.1-TOPO (Invitrogen). ΔgplHc was subsequently excised from the pCR2.1-TOPO construct using KpnI and PacI, and the excerpt was ligated to p2NIL [57] linearized by KpnI-PacI digestion. The resulting p2NIL-ΔgplHc plasmid and plasmid

pGOAL19 [57] were digested with PacI, and the PacI cassette (GOALc, 7,939 bp) of pGOAL19 was ligated to the linearized p2NIL-ΔgplHc to create p2NIL-GOALc-ΔgplHc. To create pCP0-gplH, the plasmid used for complementation analysis, a DNA fragment (266 bp) encompassing gplH and its predicted ribosome binding site (RBS) was PCR-amplified from genomic DNA with primer pair pepF and pepR and cloned into pCR2.1-TOPO. The RBS-gplH fragment was subsequently excised from the pCR2.1-TOPO construct using PstI and HindIII and ligated to plasmid pCP0 [4] linearized by PstI-HindIII mafosfamide digestion to create pCP0-gplH. The cloning placed gplH under the control of the hsp60 promoter of pCP0 for gene expression in mycobacteria. Extraction and thin layer chromatography (TLC) analysis of GPLs GPLs were extracted and analyzed by TLC by reported methods [22, 60]. Cells from cultures (5 ml, OD600 of 1.3-1.6) grown in supplemented 7H9 as described above were collected by centrifugation (4,700 × g, 15 min), washed with cold phosphate buffered saline (PBS, 1 ml), and processed for GPL extraction. GPLs were extacted with 2:1 CHCl3/CH3OH (20 μl/mg wet weight) by incubation overnight at room temperature in a rocking shaker.