Identification of an apiosyltransferase in the plant pathogen Xanthomonas pisi

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.

稀有支链糖芹糖（Apiose）曾被认为仅存在于植物界，如今却在玫瑰色球菌（Geminicoccus roseus）和豌豆黄单胞菌（Xanthomonas pisi）两种细菌中被发现。带有芹糖残基的聚糖（Glycan）可在豌豆黄单胞菌的甲醇水溶液可溶组分与不溶性沉淀组分中被检测到。研究人员已在这两种细菌中鉴定出编码细菌尿苷二磷酸芹糖（UDP-apiose）合酶（bUASs）的基因，但将芹糖整合至聚糖中的相关酶仍未明确。在豌豆黄单胞菌的基因组中，XpUAS侧翼的两个基因被注释为假定糖基转移酶（GT）蛋白。第一种糖基转移酶（本文命名为XpApiT）属于90家族糖基转移酶，具有勒洛尔B型折叠（Leloir type B）结构以及假定的脂多糖修饰（LPS）结构域；第二种糖基转移酶（本文命名为XpXylT）属于2家族糖基转移酶，具有A型折叠结构。研究人员将XpXylT与XpApiT基因克隆，并在大肠杆菌（E. coli）中进行异源表达。对携带UAS的XpXylT或XpApiT表达菌株的核苷酸糖提取物进行分析后发现，重组XpApiT以UDP-apiose为底物，而XpXylT以UDP-木糖（UDP-xylose）为底物。间接活性测定（UDP-Glo）结果表明，XpApiT是一种可特异性利用UDP-apiose的芹糖基转移酶（ApiT）。进一步的验证实验通过在大肠杆菌中共表达UAS与XpApiT完成：该共表达体系可利用UDP-apiose生成可在沉淀组分中检测到的芹糖苷（apioside），这进一步支持了XpApiT的芹糖基转移酶活性。本研究证实豌豆黄单胞菌具备合成功能尚不明确的芹糖苷的能力，但细菌芹糖基转移酶的演化机制仍有待阐明。从遗传学与进化学视角来看，芹糖操纵子（apiose operon）为探究细菌类群分化过程中基因组变化如何反映生态适应提供了独特的研究机遇。