A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

Abstract

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a beta-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the alpha-branch of mycolic acids, followed by the joining of these two branches.

Figure 1

Growth of the parent mc²-155 (▴) and mutant 155NS1 (●) in 7H9 broth medium at 30°C. Cultures were diluted 40-fold with fresh 7H9 broth supplemented with 10% Difco OADC enrichment. At times indicated, small portions of the cultures were removed and the cell density was determined by OD₆₀₀.

Figure 2

Entry of erythromycin (A) or chenodeoxycholate (B) into whole cells of mc²-155 (■) or 155NS1 (●). To 1-ml cell suspensions in 0.1 M K-phosphate buffer (pH 7.0) preincubated at 37°C, [¹⁴C]erythromycin or [¹⁴C]chenodeoxycholate was added at time zero to a final concentration of 5 μM. At various time points, 50-μl portions of the suspension were removed, filtered, and washed. The radioactivity retained on the filter was determined by scintillation counting.

Figure 3

HPLC and TLC analysis of fatty acids. (A) Fractionation of the p-bromophenacyl esters of fatty acids from the mc²-155 (solid line) or the 155NS1 mutant (dashed line) by HPLC. Cells (10 mg dry weight) were saponified and derivatized with p-bromophenacyl bromide as described in Materials and Methods. The fatty acid esters were dissolved in 100 μl of CH₂Cl₂, and 10 μl of this solution was applied to the HPLC column. Only fractions eluted between 18 and 34 min are shown. (B) TLC analysis of fatty acid methyl esters. Lanes: 1, methyl esters of mycolates purified from mc²-155; 2, esters of fatty acids from mc²-155; 3, esters of fatty acids from 155NS1. TLC was developed with hexane/ethyl acetate (9:1) as described in Materials and Methods. The weak spot underneath the mycolate ester spot in lane 2 contains mostly epoxy-mycolate, based on two-dimensional TLC analysis (not shown).

Figure 4

Proton NMR spectrum of fatty acids purified from mutant 155NS1. p-Bromophenacyl esters of fatty acids were collected from the HPLC fractions and were saponified again to remove the p-bromophenacyl group. Free fatty acids were extracted into CDCl₃ under acidic conditions and were analyzed with a 500-MHz spectrometer. We show here the spectrum of fatty acids derived from fraction B from HPLC (peak 2 of Fig. 3A) as an example. Assignment of several resonances is shown.

Figure 5

ES-MS of fatty acids from the mutant 155NS1. Fatty acid p-bromophenacyl esters from HPLC were collected and resaponified to remove the p-bromophenacyl group. The resultant free fatty acids were subjected to EM-MS analysis. We show here the spectrum of fatty acids derived from fraction C of HPLC (peaks 3–6 of Fig. 3A).

Figure 6

Electron impact–MS of fatty acids accumulated in 155NS1. A mixture of peaks 2 and 3 from Fig. 3 was analyzed. Fragmentation shown in the Inset (from ref. 19) generates the ions with m/z of 514 and 542 (see text), CO₂, and p-bromophenacylalcohol, the last compound fragmented further to produce the most prominent doublet at m/z 183/185, as also was seen in ref. .

Figure 7

Presumed pathway for the biosynthesis of the meromycolate chain of mycolic acids in M. smegmatis. The β-hydroxy fatty acids accumulated in the 155NS1 mutant are highlighted (structure VI). Although the second desaturation step here is depicted to occur at the 3 position, there currently is no strong evidence for this proposal. R′ indicates acyl carrier protein, probably -S-AcpM.