Exploration of self-resistance mechanisms in the biosynthetic cluster of Caprazamycin

01.10.2019 bis 30.09.2020

Streptomyces produce various bioactive natural products and possess resistance systems for most of these metabolites, which are co-regulated with antibiotic biosynthesis genes. Antibiotic producing microorganisms require one or more self-resistance determinants to survival during antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its export, or modify the host to render it insensitive to the molecule.
Caprazamycins are liponucleoside antibiotics which possess activity against Gram-positive bacteria, in particular against the genus Mycobacterium including M. intracellulare, M. avium and M. tuberculosis. Both, caprazamycins and the structurally related liposidomycins inhibit the enzyme MraY translocase involved in peptidoglycane cell wall biosynthesis. Both biosynthetic gene clusters contain two possible self-resistance genes, encoding for putative phosphotransferases with high sequence similarities to the wide family of tunicamycin-resistance proteins, which confers resistance to tunicamycin in a range of different bacteria. However, no tunicamycin resistance gene was yet identified within a tunicamycin gene cluster. The function of the putative phosphotransferases within the caprazamycin gene cluster therefore remains speculative at present.
Within this project, we want to elucidate the function of both phosphotransferases contained in the caprazamycin gene cluster. Knock-out experiments indicated already that only one of the phosphotransferase can be deleted as the other cannot. Why do we have two phosphotransferases in the caprazamycin and liposidomycin gene cluster? What structural moieties are phosphorylated and how does phosphorylation affect activity and transport of the compounds? An approach analyzing gene-deletion mutants, overexpressing the corresponding enzymes and biochemical investigating the function of both phosphotransferases is proposed to gain deep insight into the self-resistance mechanism of liponucleoside antibiotics. Indeed, introduction of multicopies of resistance genes probably act positively into the production of antibiotics containing efficient efflux pumps and detoxification systems for secondary metabolites. In addition, this project may help to understand future strategies to drug resistance in pathogenic bacteria.