α⁺, α, and α’ mycolates.

<p>Positive-ion mode Q-Tof mass spectrometry of 1D TLC purified MAMEs derived from <i>S. rugosus</i> (high and middle bands) or <i>S. rotundus</i> (low band). The high migrating MAMEs with longer chain length, the middle migrating MAMEs with medium chain length, or the low migration MAMEs with shorter chain length were initially designated as α⁺, α, or α’ mycolate subclasses, respectively. The most intense molecular ions, observed here as ammonium adducts, present in each subclass are indicated in bold and italics.</p

Purification and analysis of <i>Segniliparus</i> mycolates.

<p>(A) Normal phase one-dimensional thin-layer chromatography (1D TLC) of saponified and esterified lipids from <i>M. tuberculosis (M. tb)</i>, <i>S. rugosus</i> and <i>S. rotundus</i>. From left to right: Lanes 1, 2, 3, and 7 were loaded with purified keto (1), methoxy (2), alpha (3), or mixed (7) MAMEs of <i>M. tuberculosi</i>s. Lanes 4, 5, 6 were loaded with methylated saponificates containing both fatty acyl methyl esters and MAMEs derived from <i>S. rotundus</i> (4), <i>S. rugosus</i> (5), or <i>M. tuberculosis</i> (<i>M. tb</i>, 6). (B) Positive-ion mode Q-Tof ESI-MS analysis of total MAMEs derived from <i>M. tuberculosis</i> (top panels), with insets showing mass values that deduce to chemical formulas having either 3 (α) or 4 (keto, methoxy) oxygen atoms and compared with MAMEs from <i>S. rotundus</i> and <i>S. rugosus</i> (middle and bottom panels).</p

Electron micrographs.

<p>Scanning electron micrographs of <i>S. rotundus</i> (A) and <i>S. rugosus</i> (B)<b>.</b> (C) Transmission electron micrograph of <i>S. rugosus</i>. The bands are labeled as indicated. MOM stands for mycolate outer membrane and CM stands for cytoplasmic membrane.</p

Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.

<p>(A) Purified <i>S. rugosus</i> 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 <i>m/z</i> 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 (<i>m/z</i> 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of <i>S. rugosus</i> and <i>S. rotundus</i> 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 <i>S. rugosus</i> (B) and ions 339 and 367 from <i>S. rotundus</i> (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.</p