Volume 23, Issue 2, Pages 278-289 (February 2016) Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria  Sophie Burbaud, Françoise Laval, Anne Lemassu, Mamadou Daffé, Christophe Guilhot, Christian Chalut  Cell Chemical Biology  Volume 23, Issue 2, Pages 278-289 (February 2016) DOI: 10.1016/j.chembiol.2015.11.013 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 278-289DOI: (10. 1016/j. chembiol. 2015 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Distribution of the fadE5-gap-like Locus across Mycobacterial Species (A) Phylogenetic distribution of mycobacterial species containing a pks (MSMEG_0408) ortholog. The phylogenetic tree was computed as described in Supplemental Experimental Procedures. Mycobacterial species harboring a pks ortholog are presented in red and the others in black. The scale bar at the bottom of the tree indicates estimated nucleotide substitutions per site. (B) Genomic organization of the fadE5-gap-like (MSMEG_0406-MSMEG_0413) chromosomal region of M. smegmatis compared with the orthologous loci from M. abscessus ATCC19977, M. avium 104, M. phlei RIVM601174, M. gilvum Spyr1, and M. vanbaalenii PYR-1. Orthologous genes are shown by horizontal arrows of the same color. The genome organization for these strains was obtained from the genomic databases available at the NCBI Web site (http://www.ncbi.nlm.nih.gov/nucleotide). Genes are named according to the nomenclature used in these databases. For brevity, gene names have been shortened (e.g., #0939 for M. abscessus corresponds to MAB_0939). For further details, see Table S1. Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 M. smegmatis Mutants and Lipid Production Analysis (A) Genomic organization of the MSMEG_0380-MSMEG_0413 (mmpS4-gap-like) locus in the WT strain of M. smegmatis and in the PMM mutants generated in this study. Genes are represented by solid arrows. Blue arrows indicate genes involved in GPL production, black arrows genes with unknown function or involved in regulation, orange arrows genes that belong to the fadE5-gap-like region, and white arrows genes disrupted in the various PMM mutants. See also Table S2. (B) TLC analyses of total lipids extracted from the WT, PMM217, PMM240, PMM243, PMM248, PMM255, PMM249, and PMM251 mutant strains and from the PMM240, PMM243, and PMM248 complemented strains. Eluent: CHCl3/CH3OH (90:10, v/v). The spots were visualized by spraying the plates with anthrone, followed by charring. (C) TLC analyses of cellular (left panel) and surface-exposed (right panel) lipids extracted from the WT, PMM220, PMM221, and PMM222 mutant strains, and the PMM221 and PMM222 complemented strains. Eluent: CHCl3/CH3OH (90:10, v/v). The spots were visualized by spraying the plates with anthrone, followed by charring. Positions of products A and B, GPL, and trehalose dimycolate (TDM) are indicated. Note that the absence of trehalose dimycolate in surface-exposed lipid fractions is consistent with previous reports and indicates that lipid fractions were properly prepared (Ortalo-Magne et al., 1996). Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 NMR and GC-MS Analyses of Products A and B (A) NMR spectra of products A (up) and B (down): 1D 1H-NMR spectra (left part), HMBC spectra focused on carbonyl functions (medium part), and chemical shifts of 1H and 13C of glucosyl residues (table). Spectra and table are annotated in black for the two equivalent glucosyl residues of compound A (up) and in black and red, respectively, for the resonances of non-acylated and acylated residues of compound B (down). Multiplicities are annotated in parentheses: d, doublet; dd, doublet of doublet; t, triplet; m, multiplet. Green circles on HMBC spectra (middle) highlight the major correlations between the carbonyl resonance at 173 ppm and those of methylenes at 2.28–2.39 ppm (A, up) and 2.28–2.39 and 1.5 ppm (B, down). See Figure S2 for the full 2D spectra. (B) GC profiles of fatty acid methyl esters from products A (left) and B (right). Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 MS Analyses and Structures of Products A and B (A) MALDI-TOF mass spectrum of purified product B. The major ion peaks and their fatty acyl composition are indicated. (B) MALDI-TOF mass spectrum of purified product A. The portion of the mass spectrum between m/z 4,200 and 4,600 is magnified. The mass spectrum shows broad peaks composed of badly resolved isotopic peaks (due to the poor resolution in this mass range). The pseudomolecular masses of the most abundant [M + Na]+ ions present in each peak are indicated. Tentative assignments of the chemical compositions of the mass peaks are listed in Table S3. (C) Chemical structures of products A and B. The molecular masses of the various R1 and R2 acyl groups found in products A and B are indicated in parentheses. Product B contains R1 and R2 fatty acyl groups located at the 2- and 3-positions, respectively, of one of the glucosyl residues of trehalose; however, their respective location may be inverted. Likewise, the location of the R1 fatty acyl group at the 2-position of the trehalose moiety of TPP (product A) is arbitrary. Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Lipid Production in Mycobacteria Strains Harboring the TPP Locus (A) TLC analysis of lipids extracted from the M. smegmatis mc2155, PMM221 (ΔpE) M. smegmatis mutant, M. abscessus ATCC19977-S, M. chelonae CIP104535T, M. phlei DSM43239, M. gilvum Spyr1, M. vanbaalenii PYR-1, and M. avium TMC724 strains. (B) TLC analysis of lipids extracted from the M. abscessus ATCC19977-S and -R variants and the PMM253 (ΔMAB_0939) mutant strain. Eluent: CHCl3/CH3OH (90:10, v/v). The spots were visualized by spraying the plates with anthrone, followed by charring. Positions of product A and GPL are indicated. Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Schematic Representation of the Biosynthetic Pathway of TPP A detailed description of the TPP pathway is provided in the text. m = 12, 14; n = 5, 6; p = 10, 12, 14. KS, ketoacylsynthase; AT, acyltransferase; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; ACP, acyl carrier protein. Cell Chemical Biology 2016 23, 278-289DOI: (10.1016/j.chembiol.2015.11.013) Copyright © 2016 Elsevier Ltd Terms and Conditions