FIG 2.
Complementation of the BCG HIA defect with M. tuberculosis fosmid libraries identifies ppe37 as an essential gene for efficient HIA. (A) E. coli-mycobacterium integrating shuttle fosmid vector, pMycInt(apr)-sacB-FOS3, used for the construction of fosmid libraries. Features are as follows: sacB, sucrase gene for sucrose counterselection; int, mycobacterial attP-int region for stable site-specific integration into the mycobacterial genome; cos, lambda cos site to allow packaging into phage lambda particles; sopABC (parABSF), partition system of the bacterial F plasmid; incC, incompatibility region of the bacterial F plasmid; repE, replication initiation protein for the bacterial F plasmid; ori2 (oriS), single-copy origin of replication of bacterial F plasmid; oriV, inducible (with TrfA) origin of replication. (B) Genomic map of fosmid clones with enhanced HIA displaying a 65.2-kb region of the M. tuberculosis Erdman chromosome (nucleotides 2336070 to 2401279; genes, ERDMAN_2301 to ERDMAN_2368) with the location and orientation of the genomic DNA inserts from 10 unique fosmid clones with enhanced HIA (arrows labeled fosmid A through J). The 14.9-kb region that all 10 fosmids have in common is boxed. (C) Map of the 14.9-kb common region showing the five subregions (PCR A through E) tested for enhanced HIA. Only subregion E conferred enhanced HIA on BCG. (D) PPE37 protein alignment. BCG PPE37 (BCGT_1943; GenBank accession number AHM07863.1) is missing 9 amino acids (ARAWHALAA, amino acids 31 to 39; shown in red) and has one additional difference (S188I; in red) from the sequence of the M. tuberculosis PPE37 protein (ERDMAN_2339, GenBank accession number WP_003411053.1).