The nonredundant roles of two 4′-phosphopantetheinyl transferases in vital processes of Mycobacteria

Abstract

Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4 -phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4 -phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and nonribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Gué rin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs.fatty acid synthase ͉ lipid metabolism ͉ mycolic acids ͉ polyketide

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

“…These expression systems allowed the construction of conditional knockdown mutants of M. smegmatis and M. bovis BCG, in which expression of a single target gene can be silenced either by the addition (Tet-OFF system) or removal of tetracyclines (Tet-ON system) 8,10,11 . For this study we aimed to determine if Tet-ON and Tet-OFF systems also allow generating conditional knockdown mutants for virulent Mtb and if silencing of Mtb genes can be achieved during mouse infections.…”

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

“…1). Its ACP domains are naturally activated by the 4Ј-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group.…”

“…We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of ␣-alkyl ␤-ketoacids, the precursors of mycolic acids. Protein Production and Purification-The pks13 (Rv3800c) gene of M. tuberculosis H37Rv was cloned into plasmid pET26b (Novagen) upstream from a His tag coding sequence as previously described (14). The sfp gene was amplified by PCR from pUC8Sfp vector (15) and inserted into pET-26b, downstream from the T7 promoter.…”

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

“…For example, PPTase is essential for the production of mupirocin in P. fluorescens (Shields et al 2010), erythromycin in Saccharopolyspora erythraea (Weissman et al 2004), fatty acid in E. coli strain O157:H7 (Lay and Lay and Cronan 2006), both fatty acid and siderophore in P. aeruginosa (Barekzi et al 2004) and for the biosynthesis of virulence factors in Mycobacterium (Chalut et al 2006). Identification of the role of ZmsO in zeamines production adds a new member to the list of PPTases which are associated with the biosynthesis of secondary metabolites with biological significances.…”