RESUMO
Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.
Assuntos
Flagelina , Flagelina/metabolismo , Flagelina/genética , Glicosilação , Shewanella/metabolismo , Shewanella/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/genética , Evolução MolecularRESUMO
Many bacterial flagella are specifically O-glycosylated with nonulosonic acids, including the sialic acid derivatives, pseudaminic acid or legionaminic acid. Unlike protein glycosyltransferases that are extracytoplasmic, flagellin glycosyltransferases (fGTs) act cytoplasmically with unknown donor or acceptor specificities. The recent reconstitution of fGT-based glycosylation in heterologous hosts enables analyses underpinning such specificity.
Assuntos
Flagelina , Glicosiltransferases , Flagelina/genética , Glicosilação , Glicosiltransferases/metabolismo , Ácido N-AcetilneuramínicoRESUMO
On legume crops, formation of developmentally mature nodules is a prerequisite to efficient nitrogen fixation by populations of rhizobial bacteroids established inside nodule cells. Development of root nodules and concomitant microbial colonisation of plant cells are constrained by sets of recognition signals exchanged by infecting rhizobia and their legume hosts, with much of the specificity of symbiotic interactions being determined by the flavonoid cocktails released by legume roots and the strain-specific nodulation factors (NFs) secreted by rhizobia. Hence, much of Sinorhizobium fredii strain NGR234 symbiotic promiscuity was thought to stem from a family of >80 structurally diverse NFs and associated nodulation keys in the form of secreted effector proteins and rhamnose-rich surface polysaccharides. Here, we show instead that a mini-symbiotic plasmid (pMiniSym2) carrying only the nodABCIJ, nodS and nodD1 genes of NGR234 conferred promiscuous nodulation to ANU265, a derivative strain cured of the large symbiotic plasmid pNGR234a. The ANU265::pMiniSym2 transconjugant triggered nodulation responses on 12 of the 22 legumes we tested. On roots of Macroptilium atropurpureum, Leucaena leucocephala and Vigna unguiculata, ANU265::pMiniSym2 formed mature-like nodule and successfully infected nodule cells. While cowpea and siratro responded to nodule colonisation with defence responses that eventually eliminated bacteria, L. leucocephala formed leghemoglobin-containing mature-like nodules inside which the pMiniSym2 transconjugant established persistent intracellular colonies. This data shows seven nodulation genes of NGR234 suffice to trigger nodule formation on roots of many hosts and to establish chronic infections in Leucaena cells.
