RNA-seq analysis on M. bovis AF2122/97 WT, RD900 knockout and complemented strains

The Region of Difference 900 (RD900) locus contains two pknH genes (pknH1 and pknH2) flanking the tbd2 gene. Gene pknH1 and pknH2 both encodes a eukaryotic-like serine/threonine protein kinase and tbd2 encodes a potential ABC transporter. The RD900 is deleted in M. tuberculosis H37Rv and thus only one pknH gene is left. Mycobacterium bovis AF2122/97 has an intact RD900 locus with two pknH genes however none of the PknH proteins has the proline rich region as compared to PknHTB from M. tuberculosis H37Rv. To understand the function of RD900 locus in M. bovis and identify different pathways that regulated by PknH1, PknH2 or PknHTB. We generated a RD900 locus knockout mutant via the recombineering method in M. bovis AF2122/97 and then complementing the M. bovis RD900 knockout mutants with integrative plasmids, so as to allow expression of the pknH1 and pknH2 genes of M. bovis AF2122/97 or pknH gene from M. tuberculosis H37Rv. An empty integrative plasmid (MCZ) was also transformed into M. bovis AF2122/97 RD900 knockout mutants as a control. The comparative transcriptomic study by RNA-seq analysis then was done for the M. bovis AF2122/97 WT, constructed RD900 knockout and complemented strains to discover differentially expressed genes from comparison of the global gene expression profiles among these groups of strains.