Abstract
The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-β-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.
Highlights
The accumulation of DPA in the mutant cells
In the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs)6 and metabolically related lipomannan (LM) and lipoarabinomannan (LAM), for instance, it is clear that whereas the first two to three mannosylation steps of PIM occur on the cytoplasmic side of the membrane, further steps in the biosynthesis of the more highly mannosylated PIMs LM and LAM rely upon polyprenyl-phospho-mannose (PPM) and -arabinose-dependent integral membrane glycosyltransferases and take place on the periplasmic face of the membrane [1]
Relationship of Rv3789 to small multidrug resistance (SMR) Transporters—The Rv3789 gene of M. tuberculosis H37Rv is located in a cell wall biosynthetic gene cluster (Fig. 1A)
Summary
Bacterial Strains and Growth Conditions—M. tuberculosis H37Rv ATCC 25618 were grown in minimal Sauton’s medium, Middlebrook 7H11-OADC agar, and 7H9-OADC medium supplemented with 0.05% Tween 80. The replicative plasmid used to complement the knock-out mutants, pVV16-Rv3789, was constructed by PCR-amplifying the entire coding sequence of Rv3789 from M. tuberculosis H37Rv genomic DNA using primers Rv3789.1 (5Ј-gggcccgcatatgcggttcgttgtcaccggc-3Ј) and Rv3789.2 (5Ј-cccaagcttgcggatccggaagatcacggc-3Ј) and cloning this fragment into the expression vector pVV16 [19] This construct leads to the constitutive production of a C-terminal His6tagged recombinant Rv3789 protein under control of the hsp promoter. The typical reaction mixture for monitoring the incorporation of [14C]Gal from UDP-[U-14C]Galp to AG intermediates contained 1.0 Ci of UDP-[U-14C]Galp (specific activity 285 mCi mmolϪ1; GE Healthcare), 0.1 mM ATP, 0.25 mM NADH, 62.5 M UDP-GlcNAc, TDP-Rha (prepared as described in Ref. 25), cytosol (0.1 mg of proteins), membrane fraction (1.0 mg of protein), cell envelope fraction (3.0 mg of proteins), and buffer A in a final volume of 480 l. Statistical Analysis—The results are expressed as the means Ϯ S.D. and were analyzed using one-way analysis of variance followed by Tukey test to determine significant differences between samples
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