Abstract
D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.
Highlights
Mycobacteria share several defining features with Grampositive bacteria, notably a complex heteropolysaccharide, arabinogalactan, in covalent attachment to the underlying peptidoglycan through a special linker unit, a rhamnosyl-Nacetylglucosaminyl phosphate [1]
In a search for clearly identifiable functional segments, we have conducted extensive analysis of short amino acid sequences within the Emb proteins, one of which is related to a proline-rich motif involved in polysaccharide chain length regulation by several bacterial membrane proteins, such as Wzz in Shigella flexneri, a member of a family of proteins that determines chain length in lipopolysaccharide synthesis [12], and ExoP in Sinorhizobium meliloti, which regulates the synthesis of symbiotically important exopolysaccharides [13]
Topology Prediction and Sequence Analysis of the Emb Proteins—Amino acid sequences of the three proteins, EmbA, EmbB, and EmbC, from the four species Mycobacterium avium, Mycobacterium leprae, M. smegmatis, and M. tuberculosis were analyzed in detail
Summary
Sequence Analysis of Mycobacterial EmbA, EmbB, and EmbC—DNA sequence information from finished and unfinished genomes were obtained from TIGR (www.tigr.org) and NCBI (www.ncbi.nlm.nih.gov). The mutagenic primers were combined with Apa-f and Apa-r to synthesize two DNA fragments of about 600 and 330 bp for each type of point mutation, which were used in overlapping extension to generate the 933-bp fragments These final fragments were cloned into pGEM for sequence confirmation and subsequently used in replacement of the corresponding wild type ApaI fragment in pVE. The pVED279A and pVED280G constructs were created by ligation of three DNA fragments as follows: linear pVV16 digested with NdeI and HindIII; the last 2369 bp of msembC purified from pVE after digestion with NcoI and HindIII; and a fragment encoding the first 856 bp of msembC The latter was a PCR product created with a forward primer (embC-f, as above), including an NdeI site, and a reverse primer (Nco-r279 5Ј-ACG GGC CAT GGT CAG GAT GTA GCC GTC GGC GGC CGT GTT GGC G-3Ј or Nco-r280 5Ј-ACG GGC CAT GGT CAG GAT GTA GCC GCC GTC GGC CGT GTT GGC G-3Ј), including an NcoI site (underlined), and the mutation corresponding to D279A or D280G (in boldface). Digested products were analyzed by high pH anion exchange chromatography (HPAEC) as described previously [32] and compared with references to known oligosaccharides
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