Abstract
The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.
Highlights
The structure of the mycolic acids was determined in detail some time ago [5]; notably lacking in these studies was the structure of the arabinogalactan polymer, the esterification of the mycolic acids at C5 of some arabinofuranosyl residues was established [6, 7]
The pellet was resuspended in 30 ml of 80% acetone/Milli-Q water and centrifuged at 25,000 ϫ g for 20 min to remove SDS. This was again followed by two water washes and, after freeze-drying, the material obtained was used as mycolyl arabinogalactan peptidoglycan complex for further experiments
Succinyl Residues Are Present on AG Arabinan—The cell wall core, mycolyl arabinogalactan peptidoglycan complex (mAGP), was prepared from M. tuberculosis and from M. smegmatis embCϪ under non-basic and non-acidic conditions
Summary
Bacterial Cultures—M. tuberculosis (H37Rv) was grown in GAS medium and M. smegmatis (⌬embC mutant, Ref. 20) was grown in 7H9 medium (with 0.2% glycerol and 50 g/ml kanamycin), both at 37 °C. The pellet obtained was resuspended in 30 ml of Milli-Q water containing 2% SDS and stirred gently at room temperature overnight followed by centrifugation (25,000 ϫ g). The pellet was resuspended in 30 ml of Milli-Q water containing 2% SDS and stirred gently at 100 °C for 1 h This was followed by centrifuging at 25,000 ϫ g for 30 min. The pellet was resuspended in 30 ml of 80% acetone/Milli-Q water and centrifuged at 25,000 ϫ g for 20 min to remove SDS This was again followed by two water washes and, after freeze-drying, the material obtained was used as mycolyl arabinogalactan peptidoglycan complex (mAGP) for further experiments. Two-dimensional gradient-enhanced proton-detected multiple bond correlation (gHMBC) NMR spectra were acquired in absolute value mode with 512 increments and 64 signal-averaging transients per increment. The spectra were collected in suitable mass ranges corresponding to the laser power increases
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