Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

Abstract

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.

Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

“…Indeed, Liu and Mushegian (29) identified 15 members of the GT-C superfamily, representing candidates involved in the biosynthesis of cell wall related glycans and lipoglycans in M. tuberculosis. We have continued our earlier studies (9,16,19) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex in Corynebacterianeae by studying mutants of C. glutamicum and the orthologous genes and enzymes of M. tuberculosis. Herein we present Rv3805c as a new arabinofuranosyltransferase of the GT-C superfamily that is responsible for the transfer of Araf residues from DPA to the arabinan domain to form terminal ␤(1 3 2)-linked Araf residues, which marks the "end point" for AG arabinan biosynthesis before decoration with mycolic acids.…”

“…The biosynthesis of AG involves the formation of a linear galactan chain with alternating ␤(1 3 5) and ␤(1 3 6)-D-galactofuranosyl (Galf) residues of ϳ30 residues in length from the specialized "linker unit," L-Rhap-(1 3 4)-␣-D-GlcNAc (20,21). MALDI-TOF mass spectrometry (MS) analyzes of per-Omethylated AG of C. glutamicum deleted of its single arabinofuranosyltransferase, Cg-emb, revealed that the 8th, 10th, and 12th Galf residue possessed singular Araf residues (9). These specific Araf residues were recently shown to be transferred by a specialized arabinofuranosyltransferase AftA, whose gene in all Corynebacterianeae analyzed to date is adjacent to the emb cluster (19).…”

“…with respect to the deletion of Cg-emb (9) and Cg-aftA (19), which are otherwise essential in M. tuberculosis. 5 The structural basis of AG is now well defined (4,5,8); conversely, aspects of its biogenesis remained poorly resolved.…”

“…These initial Araf residues "prime" the galactan backbone for further attachment of ␣(1 3 5)-linked Araf residues. These reactions require the arabinofuranosyltransferase activities of Mt-EmbA and Mt-EmbB or Cg-Emb, which are also targets of EMB (9,13,22), to eventually result in mature AG. The Emb and AftA proteins utilize the specialized sugar donor, ␤-D-arabinofuranosyl-1-monophosphoryl-decaprenol (DPA) (23)(24)(25), and is a characteristic feature found only in Corynebacterianeae (26 -28).…”

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

“…EmbR has originally been cloned from Mycobacterium avium in the context of identifying genes that confer ethambutol resistance (13). Ethambutol, a front-line antitubercular drug, targets a set of membraneembedded arabinosyltransferases (M. avium EmbA and -B and Mtb EmbC, -A, and -B) that are involved in arabinogalactan and lipoarabinomannan biosynthesis, a critical component of the mycobacterial cell wall (13)(14)(15)(16)(17). In M. avium, the embR gene is located upstream of embA and -B, leading to the hypothesis that embR might control expression of the Emb arabinosyltransferases.…”