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.O antigens typically constitute the distal portions of lipopolysaccharides (LPS) and help determine the diverse surface characteristics of Gram-negative bacteria. These repeat unit carbohydrate polymers vary tremendously in structure and, as a family, they exhibit all known sugars and sugar modifications, linked in myriad ways forming homopolymers and heteropolymers. Control of polymer length also varies, allowing highly uniform to completely random lengths. Great diversity of O-antigen structures even within a species is well known. Moreover, O antigens of a single strain can vary according to growth and environmental conditions. One such condition is the presence of a multicellular host (5, 18, 36, 40, 42, 44).Rhizobium etli CE3 fixes nitrogen inside root nodules it incites on the common bean Phaseolus vulgaris. The O antigen of its LPS (Fig. ​(Fig.1)1) is essential for bacterial infection during development of this symbiosis (41). In addition, at least two alterations occur in the O antigen when R. etli CE3 is grown in the presence of either the host plant or plant exudates. The content of the multiply O-methylated terminal fucose is decreased (19, 44), whereas the 2-O methylation of internal fucoses (2OMeFuc) increases twofold (Fig. ​(Fig.1)1) (15, 44). In addition to the multiply O-methylated terminal fucose and 2OMeFuc, methylation occurs always on 6-deoxytalose and likely on glucuronic acid to yield 3-O-methyl-6-deoxytalose (3OMe6dTal) and methyl-esterified glucuronyl (MeGlcA) residues (Fig. ​(Fig.1)1) (22); however, the incidence of these methylations is not known to vary with growth condition. The genetics responsible for the variable O methylations and the additions of the residues they modify have not been elucidated.Open in a separate windowFIG. 1.R. etli CE3 O-antigen structure (22). The portion of the LPS conceptually defined as O antigen begins with N-acetyl-quinovosamine (QuiNAc) at the reducing end followed by a mannose (Man) residue and a fucose (Fuc) residue. Attached to this fucose is the repeating unit consisting of one fucose residue, one 3-O-methyl-6-deoxytalose residue (3OMe6dTal), and one glucuronyl methyl ester residue (MeGlcA). The sugars of the repeating unit are added sequentially exactly five times (in most molecules). An O-acetyl group is present in each of the repeating units, but its location is unknown at this time. Growth in TY culture results in one 2-O-methylfucose (2OMeFuc) per O antigen on average (22). The O-antigen backbone is capped with a 2,3-di-O-methylfucose (referred to as the terminal residue in this report) on which additional O methylation at the 4-position is variable as indicated by parentheses. Growth of the bacteria in the presence of the host plant or plant exudates induces the increase of 2-O methylation of internal fucose (2OMeFuc) residues and decreased relative amount of the terminal residue (44).Most mutations affecting the known R. etli CE3 O-antigen structure map to a 28-kb genetic cluster on the chromosome (Fig. ​(Fig.2)2) (previously referred to as lps region α 8, 19, 37, 40, 45]). Genes and mutations within this cluster previously have been given the designations lps (9) and lpe (19). Recently, the new designation wre has been sanctioned by the Bacterial Polysaccharide Gene Database for this genetic cluster and other genes specifically devoted to the R. etli CE3 O antigen, in keeping with the system of nomenclature for bacterial polysaccharide genes (47).Open in a separate windowFIG. 2.R. etli CE3 O-antigen genetic cluster. (A) The R. etli CE3 chromosomal O antigen genetic cluster spans nucleotides 784527 to 812262 of the genome sequence (28) and consists of 25 putative ORFs. ORFs relevant to the present study are enlarged, and the relative locations of mutations are indicated. White triangles indicate mutations created by insertion of antibiotic cassettes, and black triangles indicate mutations created by Tn5 mutagenesis. The strain numbers carrying these mutations are indicated above the triangles. (B) The solid bars represent the extents of R. etli CE3 DNA cloned for complementation analysis. The scale and positions match those of the lower map in panel A.Duelli et al. (19) identified a 3-kb genetic locus that is required for the presence of the 2,3-di-O-methylfucose or 2,3,4-tri-O-methylfucose at the terminus of the O antigen. Now known to be near one end of the O-antigen genetic cluster (Fig. ​(Fig.2),2), the DNA sequence reported by Duelli et al. encompasses nucleotides 807701 to 810147 of the subsequently determined genome sequence (28). Sequence and annotation of the 3-kb locus have since been revised. In place of the four open reading frames (ORFs) suggested previously (19), the current annotation predicts two ORFs: wreA and wreC (Fig. ​(Fig.2).2). The wreA ORF is predicted to encode a methyltransferase (19), but the predicted WreC polypeptide sequence matches no known methyltransferase or glycosyltransferase or any other polypeptide sequence in the database (Fig. ​(Fig.3).3). When it became clear that this locus was part of the larger O-antigen genetic cluster, the nucleotide sequence suggested that three genes contiguous to wreA also might encode functions needed for synthesis and addition of the terminal fucose. The results to be shown bore out predictions of this hypothesis.Open in a separate windowFIG. 3.Conserved domain predictions. Spanning nucleotides 804817 to 810147 of the genome sequence (28), ORFs RHE_CH00766, RHE_CH00767, RHE_CH00768, RHE_CH00769, and RHE_CH00770 were named wreB, wreD, wreF, wreA, and wreC, respectively. Previously, wreF, wreA, and wreC were referred to as nlpe2, lpeA, and nlpe1, respectively (19). ORF RHE_CH00755, spanning nucleotides 791286 to 794093, was named wreM. Predicted positions of conserved domains are indicated by amino acid positions. Abbreviations: GT, conserved glycosyltransferase domain; MT, conserved methyltransferase domain. Gray boxes indicate the predicted transmembrane domains.The gene responsible for the other conditionally variable O-antigen methylation, the 2-O methylation of internal fucose residues (2OMeFuc), had not been identified in prior published work. However, among mutants isolated by random Tn5 mutagenesis, a few had been shown to lack 2OMeFuc entirely (44). We show here that the transposon insertions were located in the bifunctional gene wreM. Furthermore, results of directed insertion mutagenesis confirm two separate enzymatic domains encoded by this gene, with the α domain being required for the 2-O methylation activity and mutation of the other domain resulting in a truncated O antigen. Mutants from the directed mutagenesis that appeared to have no LPS defects other than the lack of 2OMeFuc served as tools to assess the importance of just this structural feature in the symbiosis with P. vulgaris.