Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

ABSTRACT The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing β-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

Some bacterial flagellins are O-glycosylated on surface-exposed Serine/Threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid, and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called Motility associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, Serine/Threonine-O-linked N-Acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and Serine/Threonine-O-linked 3-Deoxy-D-manno-octulosonic acid (Ser/Thr-O-KDO), both catalysed by Geobacillus kaustophilus Maf (putative flagellin sialyltransferase) and Clostridium botulinum Maf (putative flagellin legionaminic acid transferase). We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were co-expressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. The glycosylation of G. kaustophilus flagellin with KDO, and that of C. botulinum flagellin with Neu5Ac and KDO indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.Significance StatementGlycosylation, the modification of proteins with sugars, is one of the most common post-translational modifications observed in proteins. While glycosylation is versatile, the most common forms of glycosylation are N-glycosylation, where the N atom of Asparagine is modified with a glycan, and O-glycosylation where the O atom of serine or threonine residues is modified with a glycan. Here, we report a novel type of O-glycosylation in the bacterial flagellin proteins of two Gram-positive bacteria, Geobacillus kaustophilus and Clostridium botulinum. We demonstrate for the first time that the enzyme flagellin Maf glycosyltransferase is capable of transferring the monosaccharides, N-acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid, on to serine and threonine residues of these proteins.

Mechanistic and structural studies into the biosynthesis of the bacterial sugar pseudaminic acid (Pse5Ac7Ac)

The nonulosonic acid sugar pseudaminic acid, Pse5Ac7Ac, is present on the surface of a number of human pathogens, herein we review the mechanistic and structural characterisation of the enzymes responsible for its biosynthesis in bacteria.

Genetics behind the Biosynthesis of Nonulosonic Acid-Containing Lipooligosaccharides inCampylobacter coli

ABSTRACTCampylobacter jejuniandCampylobacter coliare the most common causes of bacterial gastroenteritis in the world. Ganglioside mimicry byC. jejunilipooligosaccharide (LOS) is the triggering factor of Guillain-Barré syndrome (GBS), an acute polyneuropathy. Sialyltransferases from glycosyltransferase family 42 (GT-42) are essential for the expression of ganglioside mimics inC. jejuni. Recently, two novel GT-42 genes,cstIVandcstV, have been identified inC. coli. Despite being present in ∼11% of currently availableC. coligenomes, the biological role ofcstIVandcstVis unknown. In the present investigation, mutation studies with two strains expressing eithercstIVorcstVwere performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules usingin vitroactivity tests with recombinant GT-42 enzymes. Here we show that CstIV and CstV are involved inC. coliLOS biosynthesis. In particular,cstVis associated with LOS sialylation, whilecstIVis linked to the addition of a diacetylated nonulosonic acid residue.IMPORTANCEDespite the fact thatCampylobacter colia major foodborne pathogen, its glycobiology has been largely neglected. The genetic makeup of theC. colilipooligosaccharide biosynthesis locus was largely unknown until recently.C. coliharbors a large set of genes associated with lipooligosaccharide biosynthesis, including genes for several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide inCampylobacter jejuni. In the present study,C. coliwas found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact on our understanding ofC. coliecology, host-pathogen interaction, and pathogenesis.