Partial redundancy in the synthesis of the D-arabinose incorporated in the cell wall arabinan of Corynebacterineae

Abstract

The major cell wall carbohydrate of Corynebacterineae is arabinogalactan (AG), a branched polysaccharide that is essential for the physiology of these bacteria. Decaprenylphosphoryl-D-arabinose (DPA), the lipid donor of D-arabinofuranosyl residues of AG, is synthesized through a series of unique biosynthetic steps, the last one being the epimerization of decaprenylphosphoryl-beta-D-ribose (DPR) into DPA, which is believed to proceed via a sequential oxidation-reduction mechanism. Two proteins from Mycobacterium tuberculosis (Rv3790 and Rv3791) have been shown to catalyse this epimerization in an in vitro system. The present study addressed the exact function of these proteins through the inactivation of the corresponding orthologues in Corynebacterium glutamicum (NCgl0187 and NCgl0186, respectively) and the analysis of their in vivo effects on AG biosynthesis. We showed that NCgl0187 is essential, whereas NCgl0186 is not. Deletion of NCgl0186 led to a mutant possessing an AG that contained half the arabinose and rhamnose, and less corynomycolates linked to AG but more trehalose mycolates, compared with the parental strain. A candidate gene that may encode a protein functionally similar to NCgl0186 was identified in both C. glutamicum (NCgl1429) and M. tuberculosis (Rv2073c). While the deletion of NCgl1429 had no effect on AG biosynthesis of the mutant, the gene could complement the mycolate defect of the AG of the NCgl0186 mutant, strongly supporting the concept that the two proteins play a similar function in vivo. Consistent with this, the NCgl1429 gene appeared to be essential in the NCgl0186-inactivated mutant. A detailed bioinformatics analysis showed that NCgl1429, NCgl0186, Rv3791 and Rv2073c could constitute, with 52 other proteins belonging to the actinomycetales, a group of closely related short-chain reductases/dehydrogenases (SDRs) with atypical motifs. We propose that the epimerization of DPR to DPA involves three enzymes that catalyse two distinct steps, each being essential for the viability of the bacterial cells.

Fig. 1

The biosynthetic pathway proposed for DPA synthesis from pRpp (Mikusova et al., 2005). The isoprenoid moiety is drawn to conform to the structure of decaprenyl phosphate identified in mycobacteria. The structure of the corynebacterial equivalent is not known. Although the structure of DPX is not known with certainty this intermediate is very likely a decaprenylphosphoryl-2-keto-β-D-erythro-pentofuranose. The role of NCgl2782 (Rv3807c) in the dephosphorylation step is putative and arises from the position of the gene on the chromosome, just beside ubiA (Rv3806c), and from its annotation as a phosphatase.

Fig. 2

Comparison of the Rv3790/3791 locus within Corynebacterineae In M. tuberculosis the locus contains Rv3790, Rv3791, aftA, embC, embA and embB and 5 other ORFs, 3 of unknown function and the 2 last annotated fadD35 and transposase. Together these ORFs cover the genome sequence (Ref seq: NC_000962) between 4,234,780 and 4,254,327. In C. glutamicum the locus contains NCgl0186 (Rv3790 ortholog), NCgl0187 (Rv3791 ortholog), aftA, Cg-emb (embC ortholog) and 2 ORFs of unknown functions. These ORFs cover the genome sequence (Ref seq: NC_003450) between 201,757 and 211,759.

Fig. 3

Multiple alignments of protein sequences of NCgl0186, Rv3791, NCgl1429, Rv2073c and the deduced consensus sequence (consensus 1). Identical residues are framed and similar amino acids are in light grey. Line above the sequence shows the conserved block chosen for computational analysis. The 3 first secondary elements predicted from NCgl0186 sequence are given. Consensus 2 corresponds to the consensus sequence obtained from clustalW alignments of all the orthologs found in Corynebacterineae given in Table S2 (see supplementary material) except for MAP1819c of M. paratuberculosis that was not considered for the end of the alignment owing to its length (187 residues). Boxed residues in consensus 2 correspond to a conserved motif that may be involved in cofactor binding.