Abstract
The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.
Highlights
Mycobacterium tuberculosis and Corynebacterium diphtheriae, the etiological agents of tuberculosis and diphtheria, respectively, are notorious members of the bacterial suborder Corynebacterineae
MAG biosynthesis and structure is often studied in M. tuberculosis, Corynebacterium species have recently been used as models to understand mAG assembly
C. glutamicum mutants lacking AftA were recently used to determine that this arabinofuranoysltransferase appends three ␣(1–5)–Araf residues to the galactan to initiate arabinan biosynthesis [27]
Summary
Arabinofuranose; FP, fluorescence polarization; Galf, galactofuranose; Galp, galactopyranose; mAG, mycolyl-arabinogalactan; UGM, uridine 5Ј-diphosphate galactopyranose mutase. Acceptors with short alkyl anomeric substituents afforded only short oligomeric saccharides, whereas those with longer lipids on the acceptor afforded polysaccharide products similar to those in cells These findings led to the proposal that M. tuberculosis GlfT2 uses the acceptor lipid as a tether and that polymer length is controlled by bivalent substrate binding [19]. To compare and contrast galactan biosynthesis in different species, we examined the galactan biosynthetic enzymes from C. glutamicum and C. diphtheriae NCTC 13129, including GlfT2 and UGM. As with the M. tuberculosis GlfT2, the product polysaccharides attained from variants with amino acid changes in the proposed donor binding site are truncated These findings indicate that GlfT2 orthologs have an intrinsic ability to control polysaccharide length. Our investigations lay a foundation for dissecting the molecular details and mechanisms of galactan biosynthesis using genetic and chemical genetic tools
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