Abstract
The rare branched-chain sugar apiose, once thought to only be present in the plant kingdom, was found in two bacterial species: Geminicoccus roseus and Xanthomonas pisi. Glycans with apiose residues were detected in aqueous methanol-soluble fractions as well as in the insoluble pellet fraction of X. pisi. Genes encoding bacterial uridine diphosphate apiose (UDP-apiose) synthases (bUASs) were characterized in these bacterial species, but the enzyme(s) involved in the incorporation of the apiose into glycans remained unknown. In the X. pisi genome two genes flanking the XpUAS were annotated as hypothetical glycosyltransferase (GT) proteins. The first GT (here on named XpApiT) belongs to GT family 90 and has a Leloir type B fold and a putative lipopolysaccharide-modifying (LPS) domain. The second GT (here on XpXylT) belongs to GT family 2 and has a type A fold. The XpXylT and XpApiT genes were cloned and heterologously expressed in E. coli. Analysis of nucleotide sugar extracts from E. coli expressing XpXylT or XpApiT with UAS showed that recombinant XpApiT utilized UDP-apiose and XpXylT utilized UDP-xylose as substrate. Indirect activity assay (UDP-Glo) revealed that XpApiT is an apiosyltransferase (ApiT) able to specifically use UDP-apiose. Further support for the apiosyltransferase activity was demonstrated by in microbe co-expression of UAS and XpApiT in E. coli showing the utilization of UDP-apiose to generate an apioside detectable in the pellet fraction. This work provides evidence that X. pisi developed the ability to synthesize an apioside of indeterminate function; however, the evolution of the bacterial ApiT remains to be determined. From genetic and evolutionary perspectives, the apiose operon may provide a unique opportunity to examine how genomic changes reflect ecological adaptation during the divergence of a bacterial group.
Highlights
Bacteria produce a large array of glycan structures that are associated with the cell surface
Gram-negative bacteria produce peptidoglycan, lipopolysaccharide (LPS), capsular polysaccharides (CPS), and some N- or O- linked glycoproteins [1, 2], which have been implicated in cellular recognition of the environment and may be important for vital pathways including
We previously discovered the presence of apiose (3-C-[hydroxymethyl]-D-erythrofuranose, Api) in the soil-dwelling plant pathogen Xanthomonas pisi [20] and subsequently isolated and characterized the nucleotide sugar donor UDP-apiose and the enzyme that forms it, UDPapiose/UDP-xylose synthase (XpUAS), in this bacteria
Summary
Bacteria produce a large array of glycan structures that are associated with the cell surface. Adherence, motility, and pathogenesis [3] Synthesis of these glycans requires sugar donors, acceptor substrates, and glycosyltransferases (GTs). We previously discovered the presence of apiose (3-C-[hydroxymethyl]-D-erythrofuranose, Api) in the soil-dwelling plant pathogen Xanthomonas pisi [20] and subsequently isolated and characterized the nucleotide sugar donor UDP-apiose and the enzyme that forms it, UDPapiose/UDP-xylose synthase (XpUAS), in this bacteria. We report the first identification of a gene encoding an apiosyltransferase (ApiT) that is specific to UDP-apiose and a second GT in the same operon that uses UDP-xylose as substrate (XylT). No apiosyltransferase has previously been purified to homogeneity nor have the genes encoding this glycosyltransferase been identified This is the first report to identify apiosyltransferase activity in bacteria
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