A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

TSAI, YI TING; MONDINO, SONIA; GAGO, GABRIELA; GRAMAJO, HUGO; CABRUJA, MATÍAS; LARA, JULIA

Open Biology

Royal Society of London

Año: 2017 vol. 7

Unlike most bacteria, mycobacteria rely on the multi-domain enzymeeukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo.These metabolites are precursors of the biosynthesis of most of the lipidspresent both in the complex mycobacteria cell wall and in the storagelipids inside the cell. In order to study the role of the type I FAS systemin Mycobacterium lipid metabolism in vivo, we constructed a conditionalmutant in the fas-acpS operon of Mycobacterium smegmatis and analysed indetail the impact of reduced de novo fatty acid biosynthesis on the globalarchitecture of the cell envelope. As expected, the mutant exhibitedgrowth defect in the non-permissive condition that correlated well withthe lower expression of fas-acpS and the concomitant reduction of FAS I, confirmingthat FAS I is essential for survival. The reduction observed in FAS Iprovoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA,and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutantto keep synthesizing mycolic acids when fatty acid biosynthesis wasimpaired. A detailed comparative lipidomic analysis showed that althoughreduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis,mycolic acids were still being synthesized in the mutant, althoughwith a different relative species distribution. However, when triacylglyceroldegradation was inhibited, mycolic acid biosynthesis was significantlyreduced, suggesting that storage lipids could be an intracellular reservoirof fatty acids for the biosynthesis of complex lipids in mycobacteria. Understandingthe interaction between FAS I and the metabolic pathways that relyon FAS I products is a key step to better understand how lipid homeostasis isregulated in this microorganism and how this regulation could play a roleduring infection in pathogenic mycobacteria.1. BackgroundAll organisms that produce fatty acids (FA) do so via a repeated cycle ofreactions. The first committed step in FA biosynthesis is the carboxylation ofacetyl-CoA by acetyl-CoA carboxylase (ACC), to form the common extenderunit malonyl-CoA. This building block is subsequently condensed and reducedin an iterative fashion until the FA chain matures for use by the cell. In eukaryotes,these reactions are catalysed by a type I fatty acid synthase (FAS I), alarge multifunctional protein to which the growing chain is covalently attached[1]. By contrast, most bacteria contain a type II system (FAS II) in which eachreaction is catalysed by a discrete protein [2]. Remarkable exceptions to thisrule are members of the Corynebacterineae, which possess a eukaryotic-liketype I FAS [3]. In mycobacteria, FA biosynthesis is initiated by the& 2017