An estimated 8 million persons develop tuberculosis each year, and over 30 million people are expected to die from the disease in this decade(1) . Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that establishes an infection in oxygen-rich alveolar macrophages of the lung(2) . Mycolic acids are long chain -alkyl--hydroxy fatty acids unique to mycobacteria and related taxa and represent major components of the cell wall(3) . Mycolic acids are thought to contribute to both drug resistance and survival in the hostile intracellular environment of the macrophage by the formation of an impermeable asymmetric lipid bilayer (4) . The biosynthetic pathway for these complex lipids is also thought to be the target for several clinically useful chemotherapeutics, including isoniazid(5) . With the increasing incidence of multidrug-resistant bacilli, alternate chemotherapeutic targets are urgently needed.

Mycolic acids have been proposed to be biosynthesized via a diversion of normal fatty acid metabolism in which short chain fatty acids are extended and modified to form lipids of exceptional length(6) . Mycobacterium smegmatis synthesizes three different series of -mycolates (which lack oxygen functionalities in the meromycolate chain outside the -hydroxy acid) shown in Fig. 1(7) . The and series are full-length mycolic acids extending to an average of 78 and 79 carbons, respectively(8) . contains two cis-olefins in the meromycolate chain, while contains only a single cis-olefin and a trans-olefin with an adjacent methyl group. In addition to these three mycolates, M. smegmatis also produces a shorter ` mycolic acid, which is 64 carbons in length as well as a full-length epoxy mycolate(9) . M. tuberculosis contains only one series of -mycolic acids that averages 78-80 carbons in length(4, 10, 11) . The tubercle bacilli also produces two oxygenated mycolic acid series, ketomycolates and methoxymycolates (not shown), which are generally of lower abundance than the series(12) . Pathogenic mycobacteria cyclopropanate a majority of their mycolic acids, whereas in saprophytic organisms, this modification is unusual(3) . The functions of the various classes of mycolic acids in each of these organisms is unknown. However, we have recently shown that cyclopropanation at the distal position confers increased resistance to in vitro killing by hydrogen peroxide(13) . The present studies were initiated to expand our understanding of the relationship between mycolic acid structure and function.

Figure 1: Structure of the major mycolic acids of M. tuberculosis and M. smegmatis. M. smegmatis produces three olefinic -mycolates, which average 78-79 carbons in length in and and 64 carbons in `. The mycolic acid contains a cis-olefins in both the proximal and distal positions. The mycolic acid contains a distal cis-olefin and a proximal trans-olefin with an adjacent methyl branch. The sole -mycolic acid (4) from M. tuberculosis contains two cis-cyclopropane moieties and averages 78-80 carbons in length. The expoxy mycolic acid from M. smegmatis contains an epoxy group at the distal position and a cis-olefin at the proximal position.

We have previously reported the identification of cma1, a gene whose protein product (cyclopropane mycolic acid synthase-1, CMAS-1) ()catalyzed the introduction of a cyclopropane at the distal position in the meromycolate chain(13) . In the course of these studies, we discovered that an unannotated related sequence had been deposited in Genbank as part of the Mycobacterium leprae genome sequencing project (accession number U00018)(14) . In this paper we report that this M. leprae sequence represents a second cyclopropane synthase with a homolog in M. tuberculosis whose protein product functions distinctly from CMAS-1 to cyclopropanate the proximal cis-olefin in mycolic acid biosynthesis.

EXPERIMENTAL PROCEDURES

Materials and Strains

Cloning and Sequencing cma2

Mycolic Acid Methyl Ester (MAME) Isolation and Purification

Two-dimensional TLC Procedure

Vectors and Constructs

Differential Scanning Calorimetry

M. smegmatis cell walls were prepared as follows: M. smegmatis (250 ml A = 1.0) culture was harvested by centrifugation and resuspended in 4 ml of 1 mM phenylmethylsulfonyl fluoride. This suspension was divided equally among eight 1.5-ml tubes containing 0.5 g of 0.1-mm glass beads. These were then placed in a Mini Beadbeater-8 (BioSpec Products, Bartlesville, OK) and lysed at maximum speed for 3 min. After briefly spinning at 5,000 g, the supernatants were removed, and fresh phenylmethylsulfonyl fluoride was added (0.4 ml/tube). The pellets were again lysed for 3 min, and the beads were allowed to settle, after which the supernatants were combined (5 ml), cooled on ice, and 2.25 ml of 10% Triton X-114 (CalBiochem) was added. After mixing on ice for several minutes, cell walls were removed by centrifugation at 100,000 g for 1 h. These were washed again in fresh 2% Triton X-114 followed by an additional wash in phosphate-buffered saline to remove excess detergent. Monoclonal antibodies used to establish purity of these cell wall preparations in Western blots (Hsp60 (IT-13), the 19-kDa lipoprotein (IT-12), and the 32-kDa -antigen (IT-49)) were provided by the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases.

RESULTS

Cloning and Characterization of the cma2 Gene from M. tuberculosis

To test this hypothesis, PCR primers were designed to amplify one region of high homology and one region of low homology between the two sequences. These two probes were used sequentially to screen colony lifts of E. coli tranformed with an M. tuberculosis cosmid library(13) . Out of approximately 300 colonies, four clones were positive with the more homologous probe while only one of these was also positive with the less homologous probe. Cosmid DNA isolated from the positive clone was digested with BamHI and probed in a Southern blot with the same probes. The clone that reacted with both probes showed a strong band at 4.2 kb as well as weaker hybridization to the 7.2-kb fragment known to contain cma1(13) . Sequences homologous to cma2 were found by Southern blot analysis to be absent from the saprophytic M. smegmatis and present in pathogenic strains of Mycobacterium avium, Mycobacterium bovis BCG, and Mycobacterium marinum (data not shown).

Sequence Analysis of cma2

A comparison of the deduced amino acid sequence of CMAS-2 (the cma2 gene product) with the other known cyclopropane synthases is shown in Fig. 2. As expected, the M. tuberculosis CMAS-2 sequence is more closely related to the M. leprae CMAS-2 sequence (73% identity) than either of the CMAS-2 are to CMAS-1 from M. tuberculosis (52% identity for each). The four proteins share the highest homology in the region corresponding to the N terminus of CMAS-1 (amino acids 14-96 of CMAS-1). Interestingly, the three cyclopropane synthases (including cyclopropane fatty acid synthase from E. coli(24) ) have variable N-terminal extensions, the significance of which is unknown. Functionally, one of the most important areas of homology shared by these four proteins spans amino acids 171-179 (using the cyclopropane fatty acid synthase numbering) and corresponds to a consensus motif of (V/L)L(E/D)XGXGXG, which has been proposed to play a role in binding of the enzyme cofactor S-adenosyl-L-methionine(25) . Cysteine 354 is also absolutely conserved and has been proposed to have catalytic function(24) .

Figure 2: Amino acid comparison of the four known cyclopropane synthases. This figure shows a multiple alignment of the deduced amino acid sequence from the M. tuberculosis cma2 gene with the M. leprae cyclopropane synthase homolog, the CMAS-1 protein from M. tuberculosis(13) and the amino acid sequence of cyclopropane fatty acid synthase from E. coli(24) . The putative S-adenosyl-L-methionine binding domain (25) spans amino acids 171-179 (using cyclopropane fatty acid synthase numbering), and cysteine 354 has been proposed to have catalytic function(24) .

CMAS-2 Expression in M. smegmatis

To confirm that this transformation was due to the putative cma2 sequence as well as to improve the extent of conversion, a 1.2-kb NruI to BamHI fragment containing the cma2 reading frame was subcloned into pMH29_Hyg. pMH29_Hyg is a derivative of pMV261 that contains a hygromycin resistance marker in place of kanamycin. Hygromycin selection has much lower background and allows much faster recovery times than kanamycin for transformed mycobacteria. ()This vector also contains a synthetic MOP in place of the Hsp60 promoter region. CMAS-2 produced from this construct was capable of converting 25% of the total mycolates to the cyclopropanated type as determined by NMR.

Identification of the CMAS-2 Product and Co-expression of CMAS-1 and 2

Figure 3: Two dimensional-TLC of M. smegmatis transformed with cma-expressing constructs. [1-C]Acetate-labeled mycolic acids were purified as described under ``Experimental Procedures.'' A, M. smegmatis (pYUB18). The epoxy (e), , , and ` are marked (see Fig. 1for structures). B, M. smegmatis (pMH29H_cma1). Spot 1 is the result of cyclopropanation of the distal olefin of (13) . C, M. smegmatis (pMH29H_cma2). Spots 2 and 3 are the result of cyclopropanation of the proximal olefin of and the epoxy mycolic acid, respectively. D, M. smegmatis (pMV206H_cma1+2). Spot 4 is the dicyclopropanated mycolic acid, which is not retarded by the silver ion impregnation. Left to right development is in unmodified silica gel, while up to down represents the silver ion impregnated dimension.

Introduction of both cma genes into M. smegmatis resulted in a pronounced change in the radio-TLC profile, which contained both the CMAS individual products as well as a unique MAME, which was unaffected by silver ion impregnation (Fig. 3D). This MAME exactly co-elutes with the major -mycolic acid from M. tuberculosis (data not shown). To confirm these structural predictions, MAMEs were purified from 1 liter of M. smegmatis containing pMV206_Hyg-cma1+cma2 and separated by preparative argentation TLC. 500 MHz of H NMR analysis of spot 4 (Fig. 4A) showed that this spot had no olefinic resonances but had cyclopropane resonances ( -0.33, 0.56, and 0.65 ppm). These were present in a 4:6 ratio with terminal methyl groups, indicating that this M. smegmatis mycolate corresponded to structure 4, which is the major mycolate from M. tuberculosis (Fig. 1). The mycolate corresponding to spot 1 in Fig. 3D showed H NMR resonances (Fig. 4B) corresponding to a trans-olefin at 5.3 ppm (J = 15 Hz) as well as a doublet corresponding to an -methyl group at 0.93 ppm and cyclopropane resonances as above. The olefinic and cyclopropane resonances integrated for two and four protons, respectively; thus this mycolate corresponds to the previously described structure containing a distal cyclopropane and a methyl-branched trans-olefin. Spot 2 in Fig. 3D showed H NMR resonances (Fig. 4C) consistent with a cis-olefin at 5.36 ppm (J = 10 Hz) as well as the cis-cyclopropane resonances. This mycolate also displayed no -methyl branch and is produced by CMAS-2 alone, consistent with a proximally-cyclopropanated -mycolic acid.

Figure 4: 500 MHz Proton NMR spectra of the three cyclopropanated -mycolates produced in recombinant M. smegmatis. Spectrum A shows the dicyclopropyl mycolate corresponding to structure 4 (Fig. 1), which represents the major mycolate from M. tuberculosis. By integration, the cyclopropane resonances ( -0.33, 0.56, 0.65 ppm) are present in a ratio of 4:6 with terminal methyl groups. Spectrum B shows the mycolic acid corresponding to spot 1 in Fig. 3D, which contains a trans double bond ( 5.36 ppm, J = 15 Hz) and a single cyclopropane. By integration the olefinic and cyclopropyl protons represent two and four protons with respect to terminal methyl groups. Spectrum C shows the mycolic acid corresponding to spot 2 in Fig. 3D, which contains a single cis-olefin as well as a single cis-cyclopropane. This mycolate also lacks the -methyl branch since there is no doublet at 0.95ppm. Spectra were recorded in deuterochloroform and are referenced to internal tetramethylsilane.

Radio-TLC data for M. smegmatis transformants containing cma genes were quantitated by PhosphorImaging analysis (Table 1). Introduction of CMAS-1 decreases the series from which it is derived, while the series decreases when CMAS-2 is present. Interestingly, the total amount of mycolate cyclopropanated at the distal position in the coexpressing construct (where cma1 is expressed from its own promoter) is twice that produced when the gene is expressed from the MOP promoter. The total amount of mycolic acids cyclopropanated at the proximal position is the same between the coexpressor and the overexpressor, suggesting that CMAS-1 activity is affected by the presence of CMAS-2 but not vice versa.

Effect of Cyclopropanation on Cell Wall Fluidity

Figure 5: Differential scanning calorimetry of cell walls and purified mycolic acids from recombinant M. smegmatis. A, purified mycolic acids from M. smegmatis (pYUB18) (solid line), M. smegmatis (pYUB18_cma1) (dashed line), and M. smegmatis (pYUB18_cma2) (dotted line) were analyzed by DSC as described under ``Experimental Procedures.'' Introduction of a cyclopropane at the proximal position by CMAS-2 results in consistently higher transition temperatures than control or distally cyclopropanated (CMAS-1) material. B, Triton X-114-extracted cell wall material was prepared and analyzed by DSC. M. smegmatis (pYUB18) (solid line), M. smegmatis (pYUB18_cma2) (dotted line), M. smegmatis (pMV206_Hyg-cma1+cma2) (dashed line).

DISCUSSION

In this work, we have used a homologous sequence from the M. leprae genome sequencing project to identify the protein involved in construction of the proximal cyclopropane from M. tuberculosis. This enzyme is the fourth identified member of a family of proteins to catalyze the transfer of a methylene group from S-adenosyl-L-methionine to the double bond of a fatty acid substrate. The three mycobacterial members of this family are closely related to one another, with the cma2 genes from M. leprae and M. tuberculosis being more closely related to one another (73% identity) than to the cma1 gene of M. tuberculosis (52% identity). Heterologous expression of cma2 in M. smegmatis results in a proportion of the -mycolates becoming cyclopropanated at the proximal position. Expression of cma1 results in cyclopropanation at the distal position, while coexpression of both genes results in the production of a dicyclopropyl mycolate nearly identical to the major mycolic acid produced by M. tuberculosis.

The cyclopropanation of the epoxy mycolates by CMAS-2 in M. smegmatis suggests that the enzyme is either insensitive to substituents occurring toward the end of the chain or that CMAS-2 acts on a precursor meromycolate, which can become either cyclopropanated to form the dicyclopropyl mycolate or further oxidized to form the epoxy series. CMAS-2 activity is unchanged upon co-expression of both cyclopropane synthases with about 30% of the total mycolates cyclopropanated at the proximal position (Table 1). Total CMAS-1 activity, however, increases upon coexpression from 30 to 50% cyclopropanation of the distal position. One interpretation of this result is that the distal cyclopropane is formed after the proximal cyclopropane with CMAS-1 preferentially recognizing the proximally cyclopropanated precursor as a substrate.

The biological significance of lipid cyclopropanation has been most extensively studied in E. coli; however, the lack of any dramatic phenotype associated with either cyclopropane fatty acid synthase null mutants or cyclopropane fatty acid synthase overexpressors has left the role cyclopropanation plays in cellular metabolism unclear(28, 29) . A large increase in the synthesis of cyclopropane-containing plasma membrane fatty acids has been shown to accompany the transition from log to stationary phase, which suggests that cyclopropanation offers some protective advantage to stationary cultures(27) . E. coli, which have been grown on cyclopropane fatty acids, are more resistant to killing by hyperbaric oxygen treatment, suggesting that cyclopropanes do have a stabilizing or rigidifying effect on the membrane(30) . This is confirmed by the increased susceptibility to killing by freezing observed in cyclopropane fatty acid synthase mutants of E. coli(29) . It has also been shown by examining the H NMR of specifically deuterated cyclopropane-containing lipids, that cyclopropanated membranes enhance stability by suppressing segmental mobility of hydrocarbon chains, thus providing increased rigidity with respect to external shock(31) . These studies consistently support the position that cyclopropanation of membrane lipids, although a rather subtle modification, does contribute to increased structural integrity of membranes containing short chain fatty acids(32) . In addition, cyclopropanation is intermediate in fluidity effects between the more fluid cis-olefin and the less fluid trans-olefin as measured by DSC (33) .

Recent work on the structure of the mycobacterial cell wall suggests that the proximal cyclopropane lies at the boundary of what Minnikin (3, 26) has referred to as the structural permeability barrier. A dramatic high temperature phase transition has recently been demonstrated to occur at 60 °C in purified cell walls of M. chelonei by DSC(4) . The temperature of this transition suggests that at physiologically relevant temperatures, much of the cell wall exists in a state of exceptionally low fluidity. Cyclopropanation of mycolic acids, in addition to rendering lipids less susceptible to peroxidation, may decrease the actual fluidity even more, thus contributing to the overall impermeability of the cell wall. We examined the effect of substitution of a cis-olefin with a cis-cyclopropane in mycolic acids on cell wall thermochemistry and showed, with either purified cell walls or MAMEs, that proximal cyclopropanation increased the observed temperature of the transition by approximately 3 °C. The magnitude of this change seems quite reasonable since substitution of a cis-cyclopropane for a cis-olefin in the much shorter palmitoleate (C16:1), raises the observed temperature of phase transition by 15.6 °C(33) , and only about 30% of the mycolates are converted to the cyclopropanated form. The distal cyclopropane had no such effect, possibly reflecting the role of this cyclopropane in interacting with other lipids that form a less tightly associated region that is not observed by DSC of detergent-extracted cell walls. In fact, our M. smegmatis cell wall preparations gave significantly lower melting temperatures than purified cell walls from M. smegmatis prepared without detergent extraction ()presumably due to the loss of ancillary lipids during the Triton X-114 extraction.

The impermeability of the mycobacterial cell wall is a hallmark of the organism. In the case of slow growing and pathogenic mycobacteria such as M. tuberculosis, it seems likely that high durability of mycolic acids would be essential, especially in the face of environmental and host-initiated oxidative stress in its intracellular habitat(13, 16) . Dicyclopropyl mycolic acids are the major species found in many slow growing and pathogenic strains of mycobacteria including M. avium, Mycobacterium kansasii, M. marianum, M. leprae, Mycobacterium paratuberculosis, and M. tuberculosis(3) . In contrast fast growing saprophytic mycobacteria such as M. smegmatis, Mycobacterium phlei, and Mycobacterium chelonae appear to possess primarily diunsaturated mycolic acids with an abundance of cis-olefins(34) . In the case of the distal cyclopropane, we have previously demonstrated that expression in M. smegmatis results in significant protection from hydrogen peroxide (13) . In the case of the proximal cyclopropane, we have been unsuccessful in demonstrating a similar role in protection from oxidative stress (data not shown). This may be related to the largely internal and less accessible location of the proximal cyclopropane.

Cyclopropanation of fatty acids only occurs in a small number of related taxa of bacteria. Among mycobacteria, this modification is limited to the slow growing pathogens. Mammals do not cyclopropanate unsaturated lipids. Thus, enzymes catalyzing this unique modification constitute a viable target for the design of new chemotherapy against pathogenic mycobacteria, as well as providing the tools for understanding the biosynthesis, regulation, and function of these complex lipids.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34637[GenBank].

§

To whom correspondence should be addressed: Rocky Mountain Laboratories, 903 S. Fourth St., Hamilton, MT 59840. Tel.: 406-363-9309; Fax: 406-363-9380; kestrel@rml.niaid.pc.niaid.nih.gov.

()

The abbreviations used are: CMAS, cyclopropane mycolic acid synthase; PCR, polymerase chain reaction; MAME, mycolic acid methyl ester; TLC, thin-layer chromatography; kb, kilobase(s); MOP, mycobacterial optimal promoter; DSC, differential scanning calorimetry.

()

Y. Yuan and C. E. Barry III, unpublished results.

()

J. Liu and H. Nikaido, personal communication.

ACKNOWLEDGEMENTS

REFERENCES

1. Dolin, P. J., Ravïglione, M. C., and Kochi, A. (1994) Bull. WHO 72, 213-220 [Medline] [Order article via Infotrieve]
2. Hopewell, P. C. (1994) in Tuberculosis: Pathogenesis, Protection, and Control (Bloom, B. R., ed) pp. 25-46, American Society for Microbiology, Washington, D. C.
3. Minnikin, D. E. (1982) in The Biology of the Mycobacteria (Ratledge, C., and Stanford, J., eds) pp. 95-184, Academic Press, San Diego, CA
4. Liu, J., Rosenberg, E. Y., and Nikaido, H. (1995) Proc. Natl. Acad. Sci. U. S. A. , in press
5. Winder, F. G. (1982) in The Biology of the Mycobacteria (Ratledge, C., and Stanford, J., eds) pp. 353-438, Academic Press, San Diego, CA
6. Takayama, K., and Qureshi, N. (1984) in The Mycobacteria: A Sourcebook (Kubica, G. P., and Wayne, L. G., eds) Vol. 15, pp. 315-344, Dekker, New York
7. Wong, M. Y. H., Steck, P. A., and Gray, G. R. (1979) J. Biol. Chem. 254, 5734-5740 [Free Full Text]
8. Danielson, S. J., and Gray, G. R. (1982) J. Biol. Chem. 257, 12196-12203 [Free Full Text]
9. Wong, M. Y. H., and Gray, G. R. (1979) J. Biol. Chem. 254, 5741-5744 [Abstract/Free Full Text]
10. Qureshi, N., Takayama, K., Jordi, H. C., and Schnoes, H. K. (1978) J. Biol. Chem. 253, 5411-5417 [Abstract/Free Full Text]
11. Kaneda, K., Naito, S., Imaizumi, S., Yano, I., Mizuno, S., Tomiyasu, I., Baba, T., Kusunose, E., and Kusunose, M. (1986) J. Clin. Microbiol. 24, 1060-1070 [Abstract/Free Full Text]
12. Minnikin, D. E., and Polgar, N. (1967) J. Chem. Soc. Chem. Comm. 33, 1172-1174
13. Ying, Y., Lee, R. E., Besra, G. S., Belisle, J. T., and Barry, C. E., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6630-6634 [Abstract/Free Full Text]
14. Eiglmeier, K., Honore, N., Woods, S. A., Caudron, B., and Cole, S. T. (1993) Mol. Microbiol. 7, 197-206 [CrossRef][Medline] [Order article via Infotrieve]
15. Mathur, M., and Kolattukudy, P. E. (1992) J. Biol. Chem. 267, 19388-19395 [Abstract/Free Full Text]
16. Kennerly, D. A. (1986) J. Chromatogr. 363, 462-467 [CrossRef]
17. Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R., Kieser, T., Bloom, B. R., and Jacobs, W. R., Jr. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6987-6991 [Abstract/Free Full Text]
18. Sarmientos, P., Sylvester, J. E., Contente, S., and Cashel, M. (1983) Cell 32, 1337-1346 [CrossRef][Medline] [Order article via Infotrieve]
19. Garbe, T. R., Barathi, J., Barnini, S., Zhang, Y., Abou-Zeid, C., Tang, D., Mukherjee, R., and Young, D. B. (1994) Microbiology 140, 133-138 [Abstract]
20. Davis, E. O., Thangaraj, H. S., Brooks, P. C., and Colston, M. J. (1994) EMBO J. 13, 699-703 [Medline] [Order article via Infotrieve]
21. Sherman, D. R., Sabo, P. J., Hickey, M. J., Arain, T. M., Mahairas, G. G., Yuan, Y., Barry, C. E., III, and Stover, C. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6625-6629 [Abstract/Free Full Text]
22. Baess, I., and Mansa, B. (1978) Acta Path. Microbiol. Scand. Sect. B 86, 309-312
23. Bibb, M. J., Findlay, P. R., and Johnson, M. W. (1984) Gene (Amst.) 30, 157-166 [CrossRef][Medline] [Order article via Infotrieve]
24. Wang, A.-Y., Grogan, D. W., and Cronan, J. E., Jr. (1992) Biochemistry 31, 11020-11028 [CrossRef][Medline] [Order article via Infotrieve]
25. Ingrosso, D., Fowler, A. V., Bleibaum, J., and Clark, S. (1989) J. Biol. Chem 264, 20131-20139 [Abstract/Free Full Text]
26. Nikaido, H., Kim, S.-H., and Rosenberg, E. Y. (1993) Mol. Microbiol. 8, 1025-1030 [Medline] [Order article via Infotrieve]
27. Wang, A.-I., and Cronan, J. E., Jr. (1994) Mol. Microbiol. 11, 1009-1017 [CrossRef][Medline] [Order article via Infotrieve]
28. Grogan, D. W., and Cronan, J. E. (1984) J. Bacteriol. 158, 286-295 [Abstract/Free Full Text]
29. Grogan, D. W., and Cronan, J. E. (1986) J. Bacteriol. 166, 872-877 [Abstract/Free Full Text]
30. Harley, J. B., Santangelo, G. M., Rasmussen, H., and Goldfine, H. (1978) J. Bacteriol. 134, 808-820 [Abstract/Free Full Text]
31. Dufourc, E. J., Smith, I. C. P., and Jarrell, H. C. (1983) Chem. Phys. Lipids 33, 153-177 [CrossRef][Medline] [Order article via Infotrieve]
32. Dufourc, E. J., Smith, I. C. P., and Jarrell, H. C. (1984) Biochemistry 23, 2300-2309 [CrossRef]
33. Silvius, J. R., and McElhaney, R. N. (1979) Chem. Phys. Lipids 25, 125-134 [CrossRef]
34. Brennan, P. J., and Nikaido, H. (1995) Annu. Rev. Biochem. 64, 29-63 [CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Bhatt, N. Fujiwara, K. Bhatt, S. S. Gurcha, L. Kremer, B. Chen, J. Chan, S. A. Porcelli, K. Kobayashi, G. S. Besra, et al. Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice PNAS, March 20, 2007; 104(12): 5157 - 5162. [Abstract] [Full Text] [PDF]

				P. Ternes, P. Sperling, S. Albrecht, S. Franke, J. M. Cregg, D. Warnecke, and E. Heinz Identification of Fungal Sphingolipid C9-methyltransferases by Phylogenetic Profiling J. Biol. Chem., March 3, 2006; 281(9): 5582 - 5592. [Abstract] [Full Text] [PDF]

				F. Boissier, F. Bardou, V. Guillet, S. Uttenweiler-Joseph, M. Daffe, A. Quemard, and L. Mourey Further Insight into S-Adenosylmethionine-dependent Methyltransferases: STRUCTURAL CHARACTERIZATION OF Hma, AN ENZYME ESSENTIAL FOR THE BIOSYNTHESIS OF OXYGENATED MYCOLIC ACIDS IN MYCOBACTERIUM TUBERCULOSIS J. Biol. Chem., February 17, 2006; 281(7): 4434 - 4445. [Abstract] [Full Text] [PDF]

				G. A. Cangelosi, J. S. Do, R. Freeman, J. G. Bennett, M. Semret, and M. A. Behr The Two-Component Regulatory System mtrAB Is Required for Morphotypic Multidrug Resistance in Mycobacterium avium Antimicrob. Agents Chemother., February 1, 2006; 50(2): 461 - 468. [Abstract] [Full Text] [PDF]

				G. Etienne, F. Laval, C. Villeneuve, P. Dinadayala, A. Abouwarda, D. Zerbib, A. Galamba, and M. Daffe The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain? Microbiology, June 1, 2005; 151(6): 2075 - 2086. [Abstract] [Full Text] [PDF]

				K. Takayama, C. Wang, and G. S. Besra Pathway to Synthesis and Processing of Mycolic Acids in Mycobacterium tuberculosis Clin. Microbiol. Rev., January 1, 2005; 18(1): 81 - 101. [Abstract] [Full Text] [PDF]

				J. Starck, G. Kallenius, B.-I. Marklund, D. I. Andersson, and T. Akerlund Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions Microbiology, November 1, 2004; 150(11): 3821 - 3829. [Abstract] [Full Text] [PDF]

				P. Bandyopadhyay, H. Xiao, H. A. Coleman, A. Price-Whelan, and H. M. Steinman Icm/Dot-Independent Entry of Legionella pneumophila into Amoeba and Macrophage Hosts Infect. Immun., August 1, 2004; 72(8): 4541 - 4551. [Abstract] [Full Text] [PDF]

				D. M. Roberts, R. P. Liao, G. Wisedchaisri, W. G. J. Hol, and D. R. Sherman Two Sensor Kinases Contribute to the Hypoxic Response of Mycobacterium tuberculosis J. Biol. Chem., May 28, 2004; 279(22): 23082 - 23087. [Abstract] [Full Text] [PDF]

				N. Sung, K. Takayama, and M. T. Collins Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis Appl. Envir. Microbiol., March 1, 2004; 70(3): 1688 - 1697. [Abstract] [Full Text] [PDF]

				M. S. Glickman The mmaA2 Gene of Mycobacterium tuberculosis Encodes the Distal Cyclopropane Synthase of the alpha -Mycolic Acid J. Biol. Chem., February 28, 2003; 278(10): 7844 - 7849. [Abstract] [Full Text] [PDF]

				G. Etienne, C. Villeneuve, H. Billman-Jacobe, C. Astarie-Dequeker, M.-A. Dupont, and M. Daffe The impact of the absence of glycopeptidolipids on the ultrastructure, cell surface and cell wall properties, and phagocytosis of Mycobacterium smegmatis Microbiology, October 1, 2002; 148(10): 3089 - 3100. [Abstract] [Full Text] [PDF]

				L. Kremer, Y. Guerardel, S. S. Gurcha, C. Locht, and G. S. Besra Temperature-induced changes in the cell-wall components of Mycobacterium thermoresistibile Microbiology, October 1, 2002; 148(10): 3145 - 3154. [Abstract] [Full Text] [PDF]

				M. Watanabe, Y. Aoyagi, H. Mitome, T. Fujita, H. Naoki, M. Ridell, and D. E. Minnikin Location of functional groups in mycobacterial meromycolate chains; the recognition of new structural principles in mycolic acids Microbiology, June 1, 2002; 148(6): 1881 - 1902. [Abstract] [Full Text] [PDF]

				X. Bao, S. Katz, M. Pollard, and J. Ohlrogge Carbocyclic fatty acids in plants: Biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida PNAS, May 14, 2002; 99(10): 7172 - 7177. [Abstract] [Full Text] [PDF]

				C.-c. Huang, C. V. Smith, M. S. Glickman, W. R. Jacobs Jr., and J. C. Sacchettini Crystal Structures of Mycolic Acid Cyclopropane Synthases from Mycobacterium tuberculosis J. Biol. Chem., March 22, 2002; 277(13): 11559 - 11569. [Abstract] [Full Text] [PDF]

				V. Puech, M. Chami, A. Lemassu, M.-A. Lanéelle, B. Schiffler, P. Gounon, N. Bayan, R. Benz, and M. Daffé Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane Microbiology, May 1, 2001; 147(5): 1365 - 1382. [Abstract] [Full Text]

				A. E. DeBarber, K. Mdluli, M. Bosman, L.-G. Bekker, and C. E. Barry 3rd Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis PNAS, August 15, 2000; 97(17): 9677 - 9682. [Abstract] [Full Text] [PDF]

				M. A. Behr, B. G. Schroeder, J. N. Brinkman, R. A. Slayden, and C. E. Barry III A Point Mutation in the mma3 Gene Is Responsible for Impaired Methoxymycolic Acid Production in Mycobacterium bovis BCG Strains Obtained after 1927 J. Bacteriol., June 15, 2000; 182(12): 3394 - 3399. [Abstract] [Full Text]

				L. Wang, R. A. Slayden, C. E. Barry III, and J. Liu Cell Wall Structure of a Mutant of Mycobacterium smegmatis Defective in the Biosynthesis of Mycolic Acids J. Biol. Chem., March 15, 2000; 275(10): 7224 - 7229. [Abstract] [Full Text] [PDF]