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Ultralong C100 mycolic acids support the assignment of Segniliparus as a new bacterial genus.

Hong S, Cheng TY, Layre E, Sweet L, Young DC, Posey JE, Butler WR, Moody DB - PLoS ONE (2012)

Bottom Line: Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100.Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane.Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.

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1H NMR analysis and structural summary of segnilomycolates.(A) 1D TLC purified MAMEs were analyzed by 1H NMR. Spectral assignment for the chemical shifts and splitting patterns of mycolates were based on comparison with values reported in the literature. The large peak at 1.56 ppm was attributed to water and the singlet at 3.49 ppm was attributed to methanol in the samples. (B) Structural summary of Segniliparus mycolic acids. The number of carbons between the functional groups on the meromycolate chains were not determined here, but are based on approximate lengths previously reported.
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pone-0039017-g005: 1H NMR analysis and structural summary of segnilomycolates.(A) 1D TLC purified MAMEs were analyzed by 1H NMR. Spectral assignment for the chemical shifts and splitting patterns of mycolates were based on comparison with values reported in the literature. The large peak at 1.56 ppm was attributed to water and the singlet at 3.49 ppm was attributed to methanol in the samples. (B) Structural summary of Segniliparus mycolic acids. The number of carbons between the functional groups on the meromycolate chains were not determined here, but are based on approximate lengths previously reported.

Mentions: These initial conclusions, based on mass measurements, were further tested by 1H-NMR analysis of each class. For the high migrating S. rotundus doublet, there is overlap in chain length of the α+ and α mycolates. Therefore, these two samples are considered enriched for homogenous chain length, but are not pure class-specific mixtures (Fig. S3). Therefore, most 1H-NMR analyses was carried out on purified preparations of S. rugosus α+ and α-mycolates, as well as S. rotundus α’-mycolates (Fig. 5a). The proton resonances of mycolates were assigned based on published studies [19], [20], [21], [22]. Strong signals corresponding to methylene (CH2) protons in the mycolate chains were seen at 1.2–1.5 ppm, and the terminal methyl (CH3) protons appeared at 0.88 ppm. Characteristic protons of the Cα branched (Ha, 2.43 ppm, 1 H, m), the hydroxylated Cβ (Hb, 3.65 ppm, 1 H, m), and the terminal methyl group in ester linkage to the mycolate (3.71 ppm, 3 H, s) were identified in all five lipid samples, confirming the presence of the α-branch, β-hydroxyl and carboxylate, which define these lipids as mycolic acids.


Ultralong C100 mycolic acids support the assignment of Segniliparus as a new bacterial genus.

Hong S, Cheng TY, Layre E, Sweet L, Young DC, Posey JE, Butler WR, Moody DB - PLoS ONE (2012)

1H NMR analysis and structural summary of segnilomycolates.(A) 1D TLC purified MAMEs were analyzed by 1H NMR. Spectral assignment for the chemical shifts and splitting patterns of mycolates were based on comparison with values reported in the literature. The large peak at 1.56 ppm was attributed to water and the singlet at 3.49 ppm was attributed to methanol in the samples. (B) Structural summary of Segniliparus mycolic acids. The number of carbons between the functional groups on the meromycolate chains were not determined here, but are based on approximate lengths previously reported.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375245&req=5

pone-0039017-g005: 1H NMR analysis and structural summary of segnilomycolates.(A) 1D TLC purified MAMEs were analyzed by 1H NMR. Spectral assignment for the chemical shifts and splitting patterns of mycolates were based on comparison with values reported in the literature. The large peak at 1.56 ppm was attributed to water and the singlet at 3.49 ppm was attributed to methanol in the samples. (B) Structural summary of Segniliparus mycolic acids. The number of carbons between the functional groups on the meromycolate chains were not determined here, but are based on approximate lengths previously reported.
Mentions: These initial conclusions, based on mass measurements, were further tested by 1H-NMR analysis of each class. For the high migrating S. rotundus doublet, there is overlap in chain length of the α+ and α mycolates. Therefore, these two samples are considered enriched for homogenous chain length, but are not pure class-specific mixtures (Fig. S3). Therefore, most 1H-NMR analyses was carried out on purified preparations of S. rugosus α+ and α-mycolates, as well as S. rotundus α’-mycolates (Fig. 5a). The proton resonances of mycolates were assigned based on published studies [19], [20], [21], [22]. Strong signals corresponding to methylene (CH2) protons in the mycolate chains were seen at 1.2–1.5 ppm, and the terminal methyl (CH3) protons appeared at 0.88 ppm. Characteristic protons of the Cα branched (Ha, 2.43 ppm, 1 H, m), the hydroxylated Cβ (Hb, 3.65 ppm, 1 H, m), and the terminal methyl group in ester linkage to the mycolate (3.71 ppm, 3 H, s) were identified in all five lipid samples, confirming the presence of the α-branch, β-hydroxyl and carboxylate, which define these lipids as mycolic acids.

Bottom Line: Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100.Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane.Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.

Show MeSH
Related in: MedlinePlus