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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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Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.(A) Purified S. rugosus MAMEs found in the high migrating fraction on the TLC plate were dissolved in chloroform and methanol solution, loaded into a nanospray tip and analyzed by positive-ion mode electrospray ionization mass spectrometry with ion trapping. The ion at m/z 1368.5 [M+Na]+ corresponding to C92 mycolic acid methyl ester was subjected to CID-MS, yielding a fragment ion corresponding to a sodium adduct of the meroaldehyde chain (m/z 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of S. rugosus and S. rotundus were extracted with chloroform, diluted with methanol and analyzed by negative-ion mode electrospray ionization mass spectrometry. Both samples contain an ion at m/z 1330.3 ([M-H]−) corresponding to C92 mycolic acid, which was subjected to CID-MS analysis. The product ion 339 from S. rugosus (B) and ions 339 and 367 from S. rotundus (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.
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pone-0039017-g004: Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.(A) Purified S. rugosus MAMEs found in the high migrating fraction on the TLC plate were dissolved in chloroform and methanol solution, loaded into a nanospray tip and analyzed by positive-ion mode electrospray ionization mass spectrometry with ion trapping. The ion at m/z 1368.5 [M+Na]+ corresponding to C92 mycolic acid methyl ester was subjected to CID-MS, yielding a fragment ion corresponding to a sodium adduct of the meroaldehyde chain (m/z 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of S. rugosus and S. rotundus were extracted with chloroform, diluted with methanol and analyzed by negative-ion mode electrospray ionization mass spectrometry. Both samples contain an ion at m/z 1330.3 ([M-H]−) corresponding to C92 mycolic acid, which was subjected to CID-MS analysis. The product ion 339 from S. rugosus (B) and ions 339 and 367 from S. rotundus (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.

Mentions: To confirm the length of the alpha branch, we carried out nanospray MS with collision-induced dissociation (CID) analysis (Fig. 4). In the positive-ion mode analysis, we found that ions corresponding to sodium adducts [M+Na]+ of C92 and C94 MAMEs from the S. rugosus α+ fraction, as well as C78 MAMEs, each generated a fragment of m/z 354 (Fig. 4a and data not shown). This fragment is attributed to a C22∶0 fatty methyl ester, which was cleaved at the C2–C3 position [22]. Because the product ions, including the meroaldehyde chain, are not always stable in the positive-ion mode, we sought to improve sensitivity by carrying out similar experiments on free mycolic acids (MA) in the negative ion mode [23], [24]. In both species, we isolated ions at m/z 1386 (C96 MA), 1330 (C92 MA), and 1136 (C78 MA), which were deduced as [M-H]− of C96 MA, C92 MA and C78 MA. Additionally, for S. rotundus, we detected m/z 969, which was deduced as C66 MA. Each of these ions was subjected to CID-MS analysis. S. rugosus mycolates all generated single product ion 339 and no detectable chain length series (Fig. 4b). In contrast, ions 339 and 367 were found in S. rotundus (Fig. 4c). We interpret these patterns as α-chain derived fatty acyl chains of C22∶0 in S. rugosus, but C22∶0 and C24∶0 in S.rotundus. These results are in agreement with a prior pyrolysis analysis [10].


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)

Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.(A) Purified S. rugosus MAMEs found in the high migrating fraction on the TLC plate were dissolved in chloroform and methanol solution, loaded into a nanospray tip and analyzed by positive-ion mode electrospray ionization mass spectrometry with ion trapping. The ion at m/z 1368.5 [M+Na]+ corresponding to C92 mycolic acid methyl ester was subjected to CID-MS, yielding a fragment ion corresponding to a sodium adduct of the meroaldehyde chain (m/z 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of S. rugosus and S. rotundus were extracted with chloroform, diluted with methanol and analyzed by negative-ion mode electrospray ionization mass spectrometry. Both samples contain an ion at m/z 1330.3 ([M-H]−) corresponding to C92 mycolic acid, which was subjected to CID-MS analysis. The product ion 339 from S. rugosus (B) and ions 339 and 367 from S. rotundus (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375245&req=5

pone-0039017-g004: Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.(A) Purified S. rugosus MAMEs found in the high migrating fraction on the TLC plate were dissolved in chloroform and methanol solution, loaded into a nanospray tip and analyzed by positive-ion mode electrospray ionization mass spectrometry with ion trapping. The ion at m/z 1368.5 [M+Na]+ corresponding to C92 mycolic acid methyl ester was subjected to CID-MS, yielding a fragment ion corresponding to a sodium adduct of the meroaldehyde chain (m/z 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of S. rugosus and S. rotundus were extracted with chloroform, diluted with methanol and analyzed by negative-ion mode electrospray ionization mass spectrometry. Both samples contain an ion at m/z 1330.3 ([M-H]−) corresponding to C92 mycolic acid, which was subjected to CID-MS analysis. The product ion 339 from S. rugosus (B) and ions 339 and 367 from S. rotundus (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.
Mentions: To confirm the length of the alpha branch, we carried out nanospray MS with collision-induced dissociation (CID) analysis (Fig. 4). In the positive-ion mode analysis, we found that ions corresponding to sodium adducts [M+Na]+ of C92 and C94 MAMEs from the S. rugosus α+ fraction, as well as C78 MAMEs, each generated a fragment of m/z 354 (Fig. 4a and data not shown). This fragment is attributed to a C22∶0 fatty methyl ester, which was cleaved at the C2–C3 position [22]. Because the product ions, including the meroaldehyde chain, are not always stable in the positive-ion mode, we sought to improve sensitivity by carrying out similar experiments on free mycolic acids (MA) in the negative ion mode [23], [24]. In both species, we isolated ions at m/z 1386 (C96 MA), 1330 (C92 MA), and 1136 (C78 MA), which were deduced as [M-H]− of C96 MA, C92 MA and C78 MA. Additionally, for S. rotundus, we detected m/z 969, which was deduced as C66 MA. Each of these ions was subjected to CID-MS analysis. S. rugosus mycolates all generated single product ion 339 and no detectable chain length series (Fig. 4b). In contrast, ions 339 and 367 were found in S. rotundus (Fig. 4c). We interpret these patterns as α-chain derived fatty acyl chains of C22∶0 in S. rugosus, but C22∶0 and C24∶0 in S.rotundus. These results are in agreement with a prior pyrolysis analysis [10].

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