Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

Abstract

Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

Extracytoplasmic function sigma factor σD confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum

“…PorA and PorH are modified with MA by the mycolyltransferase Cmt1 (Huc et al ., ), which was also identified as the σ D regulon. The third protein modified by Cmt1 has been recently identified as a short protein encoded by cgR_2501 (cg2875 in ATCC 13032) (Issa et al ., ). Intriguingly, both rsdA deletion and sigD overexpression resulted in cgR_2501 upregulation (Supporting Information Tables S3 and S5).…”

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

“…In another example, O‐AlkTMM‐C7 was shown to label mycomembrane‐resident O‐mycoloylated proteins in Cglut (via the pathway shown in Figure A), providing a chemical biology approach to the discovery and characterization of this distinctive class of proteins . In the context of studying this post‐translational modification, the ability of O‐AlkTMM‐C7 to exclusively label O‐mycoloylated proteins provides a substantial advantage over using alkynyl fatty acids, which label all types of lipidated proteins in bacteria and thus lack selectivity for O‐mycoloylation . Overall, O‐AlkTMM‐C7 is a powerful new tool for studying various aspects of the mycomembrane.…”

The C‐terminal domain of Corynebacterium glutamicum mycoloyltransferase A is composed of five repeated motifs involved in cell wall binding and stability

“…However, such a role for the have not yet been biochemically characterized, but recent studies suggest that MytA could be modified posttranslationally by one or several mycoloyl groups (Kavunja et al, 2016). These groups may help anchor the protein in the mycomembrane, as was recently shown for PorB and PorC proteins (Carel et al, 2017;Issa et al, 2017). This may explain why the ratio between secreted and cell wall MytA is not modified in the case of the MytAhis 44-405 variant (data not shown).…”

“…Once in the envelope, mycolate chains are further transferred from TMM to final acceptors, that is, another TMM, to form trehalose di-mycolate (TDM) or, alternatively, to the terminal arabinosyl residues of the AG. Interestingly, mycolic chains can also be transferred posttranslationally onto a serine residue in porins of Corynebacterium glutamicum (Carel et al, 2017;Huc et al, 2010Huc et al, , 2013Issa et al, 2017). All these reactions involving transfer of a mycoloyl group are catalyzed by a family of enzymes collectively called mycoloyltransferases (Myts) (Dautin et al, 2017).…”