Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul;192(14):3661-8.
doi: 10.1128/JB.00312-10. Epub 2010 May 14.

Redundant function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis cyclopropanation of oxygenated mycolates

Daniel Barkan  1 , Vivek Rao, George D Sukenick, Michael S Glickman
Affiliations

Redundant function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis cyclopropanation of oxygenated mycolates

Daniel Barkan et al. J Bacteriol. 2010 Jul.

Abstract

The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Chemical structures of the major mycolic acids of M. tuberculosis. Cyclopropane rings and methyl branches are shown and annotated with the methyltransferase responsible for their synthesis.
FIG. 2.
FIG. 2.
Construction of M. tuberculosis ΔcmaA1 ΔmmaA2, ΔcmaA2 ΔmmaA2, and ΔmmaA1 strains. (A) Deletion of cmaA1 from MGM104 (ΔmmaA2). When probed with the 3′ flank of cmaA1, BglII-digested genomic DNA produces a 4.9-kb band in wild-type cells whereas the ΔcmaA1 strain has a 7.2-kb band. Two clones were also confirmed by XhoI digestion, where the WT strain is predicted to have an 11.7-kb band and the ΔcmaA1 strain is predicted to have a 4.4-kb band. (B) Deletion of cmaA2 from MGM104. When probed with the 3′ flank of cmaA2, HindIII-digested genomic DNA produces a 16.4-kb band in wild-type cells whereas the ΔcmaA2 strain is predicted to have a 3.5-kb band. (C) Deletion of mmaA1 from the WT background. When probed with the 5′ flank of mmaA1, SmaI-digested genomic DNA produces a 1.3-kb band in wild-type cells whereas the ΔmmaA1 strain is predicted to have a 3.1-kb band. EcoRI-digested genomic DNA produces 6.4- and 3.1-kb bands for the wild-type and ΔmmaA1 strains, respectively.
FIG. 3.
FIG. 3.
CmaA2 and MmaA2 are redundant for cis cyclopropanation of the oxygenated mycolates. Shown is two-dimensional argentation TLC of mycolic acid methyl esters from the M. tuberculosis wild type (A) or the ΔmmaA2 (B), ΔcmaA2 (C), or ΔcmaA1 (D) strain. Panel E shows mycolates from the ΔcmaA2 ΔmmaA2 strain (MGM1974), whereas panel F shows the ΔcmaA1 ΔmmaA2 strain (MGM1967). In panels B, E, and F, the arrow marks the position of the mature alpha-mycolates and the arrowhead marks mature methoxymycolate. In panel B, the asterisk indicates monounsaturated alpha-mycolate. In panel C, the double asterisk indicates cis-unsaturated methoxymycolates and the single asterisk indicates trans-unsaturated methoxymycolates.
FIG. 4.
FIG. 4.
Structural analysis of methoxy- and ketomycolates from the ΔcmaA2 ΔmmaA2 strain. NMR analysis of purified methoxymycolate (A) or ketomycolate (B) from WT M. tuberculosis or the ΔcmaA2 ΔmmaA2 double mutant. The relative peak area of the cis cyclopropane peak at −0.33 is indicated below each spectrum and is set to 1.0 for the wild type. Spectra are normalized to the resonance at 0.86 (equal for the WT and the mutant), representing the terminal methyl groups common to all mycolic acids. Note that the ΔcmaA2 ΔmmaA2 strain also lacks trans cyclopropyl protons due to the cmaA2 deletion, as previously reported (9).
FIG. 5.
FIG. 5.
mmaA1 is required for trans cyclopropane synthesis. (A) Two-dimensional TLC analysis of total mycolic acids from WT M. tuberculosis, the ΔmmaA1 mutant, and the complemented strain. The full-spectrum NMRs of total mycolic acids from each strain are shown immediately to the right of each corresponding TLC. (B) Magnification of the NMR spectra for the area between 0.7 and −0.5 ppm, showing the disappearance of the trans peaks at 0.15 and 0.45 in the mutant and their restoration in the complemented strain.
FIG. 6.
FIG. 6.
Loss of mmaA1 enhances cording in M. tuberculosis. Auramine-rhodamine-stained mycobacteria grown in the presence of the indicated concentrations of Tween 80 were observed by fluorescence microscopy. Representative areas of growth of WT and ΔmmaA1 M. tuberculosis are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alahari, A., L. Alibaud, X. Trivelli, R. Gupta, G. Lamichhane, R. C. Reynolds, W. R. Bishai, Y. Guerardel, and L. Kremer. 2009. Mycolic acid methyltransferase, MmaA4, is necessary for thiacetazone susceptibility in Mycobacterium tuberculosis. Mol. Microbiol. 71:1263-1277. - PubMed
    1. Barkan, D., Z. Liu, J. C. Sacchettini, and M. S. Glickman. 2009. Mycolic acid cyclopropanation is essential for viability, drug resistance, and cell wall integrity of Mycobacterium tuberculosis. Chem. Biol. 16:499-509. - PMC - PubMed
    1. Barry, C. E., III, R. E. Lee, K. Mdluli, A. E. Sampson, B. G. Schroeder, R. A. Slayden, and Y. Yuan. 1998. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res. 37:143-179. - PubMed
    1. Behr, M. A., B. G. Schroeder, J. N. Brinkman, R. A. Slayden, and C. E. Barry III. 2000. A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927. J. Bacteriol. 182:3394-3399. - PMC - PubMed
    1. Bhatt, A., N. Fujiwara, K. Bhatt, S. S. Gurcha, L. Kremer, B. Chen, J. Chan, S. A. Porcelli, K. Kobayashi, G. S. Besra, and W. R. Jacobs, Jr. 2007. Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc. Natl. Acad. Sci. U. S. A. 104:5157-5162. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources