Mycobacterium tuberculosis H37Rv, espL deletion mutant
Mycobacterium tuberculosis strains were grown at 37C in 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Middlebrook) or on 7H10 plates supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase (OADC, Middlebrook). Sautons liquid medium was used for culture filtrate analysis. Streptomycin (20 g/ml), kanamycin (20 g/ml), hygromycin (50 g/ml) or 2.5% sucrose were added when necessary. One kb up- and downstream regions of the espL gene were PCR-amplified, ligated in-frame with the AvrII site and cloned into the PacI and AscI sites of pJG1100, resulting in the suicide vector pJG1100-espL-UP/DOWN. The complementing plasmid pGA44-espL was constructed by cloning the espL gene into vector pGA44, under control of the PTR promoter. Deletion of the full-length espL gene was achieved by homologous recombination using plasmid pJG1100-espL-UP/DOWN. After transformation of M. tuberculosis H37Rv, the first recombination event was selected on 7H10 plates, supplemented with hygromycin and kanamycin. Colonies were screened by colony PCR. Two clones that had undergone homologous recombination were grown in liquid 7H9 medium with no antibiotics, in order to promote the second recombination event and plasmid excision. Selection of the recombinant clones was performed by plating the bacteria on 7H10 plates supplemented with sucrose. Libraries were prepared using the Kapa LTP Library Prep kit (Kapa Biosystems) according to the manufacturers recommendations. Cluster generation was performed using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were demultiplexed using the bcl2fastq Conversion Software.