Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis

Abstract

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

FIG. 1.

Structure of phthiocerol dimycocerosates and related compounds in M. tuberculosis. R₂ is CH₃ or C₂H₅; R₃ is (CH₂)_16-18-CH₃. Arrows show the position of the double bond reduced by the Rv2953 protein during phthiocerol and phenolphthiocerol biosynthesis.

FIG. 2.

Construction and characterization of the M. tuberculosis H37Rv Rv2953::Km mutant strain. (A) Schematic diagram of the genomic organization of the Rv2953 locus in the wild-type strain of M. tuberculosis H37Rv and the PMM80 (Rv2953::Km) mutant. The black box represents the Rv2953 gene, and the hatched box represents the fragment deleted during the construction of the knockout mutant. The Km resistance cassette used for targeted disruption is represented by a gray box. Names of primers are indicated by letters (A, 2953A; B, 2953B; C, 2953C; D, 2953D; E, 2953E; res1; and res2). Positions are indicated by arrowheads below each genetic structure, and the expected sizes for PCR products are indicated. (B) PCR analysis of the recombinant strain PMM80 using various combinations of specific primers.

FIG. 3.

TLC analyses of lipids extracted from M. tuberculosis H37Rv and its isogenic Rv2953::Km mutant strain (PMM80). (A) TLC analysis of DIM from the M. tuberculosis wild-type strain, the PMM80 mutant, and the PMM80::pC2953 strain. Lipid extracts dissolved in CHCl₃ were separated with petroleum ether-diethylether (90:10, vol/vol), and DIM were visualized by spraying the TLC plate with 10% phosphomolybdic acid in ethanol, followed by heating. Positions of DIM A and DIM B (arrows) and of products A and B (arrowheads) are indicated. (B) TLC analysis of glycolipids extracted from the M. tuberculosis wild-type and PMM80 mutant strains complemented with pPET1. Lipids were dissolved in CHCl₃ and run in CHCl₃/CH₃OH (95:5, vol/vol). Glycoconjugates were visualized by spraying the TLC plate with 0.2% anthrone (wt/vol) in concentrated H₂SO₄, followed by heating. Positions of M. tuberculosis PGL (PGL-tb; arrow) and product C (arrowhead) are indicated.

FIG. 4.

MALDI-TOF mass spectra of purified product A from the PMM80 mutant strain (panel A), of purified DIM A from M. tuberculosis wild-type H37Rv (panel B), of purified glycolipid (product C) from the PMM80::pPET1 strain (panel C), and of purified PGL from M. tuberculosis H37Rv::pPET1 (PGL-tb; panel D).

FIG. 5.

The ¹H-NMR spectrum of product A from the PMM80 mutant strain. The structure of the analyzed compound deduced from the various structural analyses is shown above the spectrum (p, p' = 3 to 5; n, n' = 16 to 18; m = 20 to 22). Letters (a to h) shown above the formula indicate the protons, and those above the spectrum indicate the corresponding signal resonances.