A molecular genetic investigation of pivotal ABC-transporters involved in mycobacterial cell envelope biosynthesis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 1.6 million deaths per annum. Mtb has a complex cell envelope that contains unique polysaccharide structures that include arabinogalactan, phosphatidyl-myo-inositol mannosides (PIMs), lipomannan and lipoarabinomannan. These glycoconjugates appear throughout Corynebacteria mycobacteria nocardia (CMN) group of bacterial organisms. However, the mechanism by which these key cell envelope components are translocated across the cytoplasmic membrane, remain to be elucidated. The ABC transporter superfamily of proteins are responsible for the movement of a wide variety of molecular species across membranes and require hydrolysis of ATP to provide energy to facilitate movement. Here I investigated two ABC transporter systems involved in galactan and PIM translocation in Corynebacteriaceae. Using phenotypic analysis of deletion mutants in C. glutamicum, I provide evidence to support that Rv1272c/Rv1273c are directly involved in PIM translocation as the deletion of the C. glutamicum orthologs results in a depletion of PIM species on the cell surface. Through characterising protein-ligand interactions between the M. smegmatis Rv3781 ortholog and immature galactan analogues, I present evidence to support the prediction that Rv3781/Rv3783 may be involved in galactan translocation. Finally, this study explores the effect of point mutations linked to Ethambutol (EMB) resistance in the key arabinosyltransferase (Emb) on the C. glutamicum cell envelope, through phenotypic characterisation of the effect on component cell envelope compounds