AftD functions as an α1 → 5 arabinofuranosyltransferase involved in the biosynthesis of the mycobacterial cell wall core

Abstract

Arabinogalactan (AG) is an essential structural macromolecule present in the cell wall of Mycobacterium tuberculosis, serving to connect peptidoglycan with the outer mycolic acid layer. The D-arabinan segment is a highly branched component of AG and is assembled in a step-wise fashion by a variety of arabinofuranosyltransferases (AraT). We have previously used Corynebacterium glutamicum as a model organism to study these complex processes which are otherwise essential in mycobacteria. In order to further our understanding of the molecular basis of AG assembly, we investigated the role of a fourth AraT, now termed AftD by generating single (ΔaftD) and double deletion (ΔaftB ΔaftD) mutants of C. glutamicum. We demonstrate that AftD functions as an α(1 → 5) AraT and reveal the point at which it exerts its activity in the AG biosynthetic pathway.

Keywords: Arabinogalactan; Cell wall; Corynebacterium glutamicum; Glycosyltransferase; Mycobacterium tuberculosis.

Fig. 1A

Common glycosyl linkage motifs found within mycobacteria and corynebacteria (A), topology of AftD (B), knock out strategy (C) and effect on growth in liquid media (D). A, The hexaarabinosyl (motif) 1 is the site of mycolic acid esterification, the [α-D-Araf(1 → 5]-α-D-Araf (motif 2) represents the main linear segments of D-arabinan, the [α-D-Araf(1 → ]3,5-α-D-Araf (motif 2) illustrates the main bifurcation points of a single D-arabinan chain, and motifs 4 and 5 show the linkage profiles of linear D-galactan configuration of how D-arabinan is attached to D-galactan. B, topology of C. glutamicum AftD with the black triangle indicating the location of the 388 aa insertion in the M. tuberculosis ortholog of AftD. The topology prediction is based on the dense alignment surface method. The conserved aspartyl and glutamyl residues are indicated as D and E, respectively. The star indicates the two catalytic motifs resembling those of glycosyltransferases of the GT-C family (Liu and Mushegian, 2003). C, strategy to construct C. glutamicumΔaftD. Shown is the wild type genomic aftD-region and the deletion vector pK19mobsacBΔaftD carrying 18 nucleotides of the 5′-end of aftD and 36 nucleotides of its 3′-end thereby enabling the in-frame deletion of almost the entire Cg-aftD gene. Selection for homologous recombination results in C. glutamicumΔaftD (Cg-aftD). The arrows marked P1 and P2 locate the primers used for the PCR analysis to confirm the absence of Cg-aftD. Distances are not drawn to scale. The results of the PCR analysis are shown on the right, where the amplification product obtained from the wild type (WT) and that of the deletion mutant of (Δ) was marked accordingly. The sizes of 4032 bp for the wild type and of 1051 bp for the deletion mutant were as expected and marked by an arrow head. St marks the standard. D, the consequences of Cg-aftD deletion and Cg-aftB/Cg-aftD double deletion on growth in rich medium (BHI). Growth of C. glutamicum (●), C. glutamicumΔaftD (■), C. glutamicumΔaftD (▲) and C. glutamicumΔaftBΔaftD (Δ).

Fig. 2

Gas Chromatography/Mass Spectrometry (GC/MS) analysis of partially per-O-methylated, per-O-acetylated alditol acetate derivatives of purified arabinogalactan from C. glutamicum, C. glutamicumΔaftB, C. glutamicumΔaftD and C. glutamicumΔaftBΔaftD. A, gas chromatograms demonstrating the presence of glycosyl linkages in purified AG. B, mass spectra fragmentation profile of peaks resolving at 11.3 min corresponding to 2-Araf (^*) and 3-Araf (^**) and cleavage ions representing fragmentation of 2-Araf (^*) and 3-Araf (^*) located in terminal arabinan glycosyl motifs.

Fig. 3

Arabinofuranosyltransferase activity utilizing neoglycolipid acceptor A and membranes prepared from C. glutamicum, C. glutamicumΔaftB, C. glutamicumΔaftD and C. glutamicumΔaftBΔaftD. A, Arabinofuranosyltransferase activity was determined using the synthetic neoglycolipid acceptor α-D-Araf(1 → 3)-α-D-Araf-O-(CH₂)₇CH₃ (acceptor A) in a cell-free assay with and without EMB (100 μg/ml) as previously described (Lee et al., 1997). The products of the assay were resuspended prior to scintillation counting (10%) and the remaining subjected to TLC using silica gel plates (5735 silica gel 60F₂₅₄, Merck) in isopropanol:acetic acid:water (8:1:1, v/v/v) with the reaction products visualized by autoradiography. The TLC autoradiogram is representative of several independent experiments. B, biosynthetic reaction scheme of products A¹, A² and A³ formed in arabinofuranosyltransferase assays using the neoglycolipid acceptor A. C, GC/MS analysis of the partially per-O-methylated, per-O-acetylated alditol acetate derivative of reaction products obtained from assays containing membranes prepared from either C. glutamicum, C. glutamicumΔaftB, C. glutamicumΔaftD and C. glutamicumΔaftBΔaftD.

Fig. 4

Arabinofuranosyltransferase activity utilizing neoglycolipid acceptor B and membranes prepared from C. glutamicum, C. glutamicumΔaftB, C. glutamicumΔaftD and C. glutamicumΔaftBΔaftD. A, Arabinofuranosyltransferase activity was determined using the synthetic neoglycolipid acceptor [α-D-Araf(1 → ]3,5-α-D-Araf-O-(CH₂)₇CH₃ (acceptor B) in a cell-free assay with and without EMB (100 μg/ml) as previously described (Lee et al., 1997). The products of the assay were resuspended prior to scintillation counting (10%) and the remaining subjected to TLC using silica gel plates (5735 silica gel 60F₂₅₄, Merck) in isopropanol:acetic acid:water (8:1:1, v/v/v) with the reaction products visualized by autoradiography. The TLC autoradiogram is representative of several independent experiments. B, Biosynthetic reaction scheme of products B¹, B² and B³ formed in arabinofuranosyltransferase assays using the neoglycolipid acceptor B. C, GC/MS analysis of the partially per-O-methylated, per-O-acetylated alditol acetate derivative of reaction products obtained from assays containing membranes prepared from either C. glutamicum, C. glutamicumΔaftB, C. glutamicumΔaftD and C. glutamicumΔaftBΔaftD.

Fig. 5

GC/MS and ES-MS characterisation of in vitro synthesized reaction products from the arabinofuranosyltransferase assays utilizing both acceptors A and B. A, GC/MS analysis and B, ES-MS analysis of products derived from assays using acceptor A and membranes prepared from C. glutamicumΔaftBΔaftD over-expressing Mt-aftD supplemented with EMB. C, GC/MS analysis and D, ES-MS analysis of products derived from assays using acceptor B and membranes prepared from C. glutamicumΔaftBΔaftD over-expressing Mt-aftD supplemented with EMB.

Fig. 6

Revised pathway of arabinan biosynthesis illustrating the individual roles of Emb, AftA, AftB, AftC and AftD arabinofuranosyltransferases.