Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB

Genetic Basis for \u3cem\u3eRhizobium etli\u3c/em\u3e CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis

Biosynthesis of the Rhizobium etli CE3 O Antigen

Canonical Gram-negative bacteria have outer membranes abundant in lipopolysaccharide (LPS). The structure of most LPSs can be discussed in terms of three general regions: the lipid A membrane anchor, the core oligosaccharide, and the distal polysaccharide O antigen. Chemical analysis shows the Rhizobium etli CE3 O antigen to be a fixed-length heteropolymer, with two O-methyl residues that vary according to growth conditions. Prior genetic analysis has identified regions within the genome necessary for O-antigen synthesis in this bacterial strain, and the predicted functions of the open reading frames (ORFs) in these regions can account for nearly all the enzymes thought to be required for the synthesis of the known O-antigen structure. Which genes are required for which predicted steps in the synthesis of the R. etli CE3 O antigen were investigated in this study. The LPS of mutants defective in these ORFs was analyzed. On high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, most mutants synthesized truncated LPSs of varying sizes and abundance. The presence or absence of specific O-antigen sugars in these truncated LPSs was investigated by sugar composition analysis. From these analyses, a model for the synthesis of the R. etli CE3 O antigen is proposed. This model defines N-acetyl-quinovosamine as the first sugar of the O antigen and, beginning with this sugar, correlates almost every gene within the Oantigen genetic clusters to a specific function in the synthesis of the O antigen. These functions include: eight predicted glycosyltransferases for the addition of each sugar in the known O-antigen structure; enzymes for synthesis of most of the O-antigen-specific sugar nucleotides, including those for N-acetyl-quinovosamine, fucose, 3-O-methyl-6- deoxytalose, and the variable multiply-O-methylated terminal fucose; and the machinery for transport of the O antigen across the inner membrane. Investigations were initiated to identify, to isolate, and to biosynthesize CE3 Oantigen intermediates. Particular combinations of genetic defects were constructed to test specific predictions of the O-antigen synthesis model. These mutants coupled with several approaches involving radiolabeling in vivo and in extracts in vitro were used to identify candidates for O-antigen intermediates in R. etli CE3

Genetic Basis for Rhizobium etli CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions▿

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis