Genetic and chemical characterization of a mutant that disrupts synthesis of the lipopolysaccharide core tetrasaccharide in Rhizobium leguminosarum

Abstract

A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced. Two open reading frames (ORFs) were identified. The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis. ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases. Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-deoxy-Dmanno-2-octulosonic acid (Kdo) residue which normally attaches the core tetrasaccharide to the O chain was missing, suggesting that lpcB may encode a CMP-Kdo:LPS Kdo transferase.The lipopolysaccharides (LPSs) of gram-negative bacteria are a major chemical constituent of the outer membrane and are implicated as having a role in infection of the legume symbiont by rhizobia (3, 10, 11). In determinate bean nodules, LPS rough mutants of Rhizobium leguminosarum bv. phaseoli accumulate in the infection thread without forming bacteroids, and nitrogen fixation does not occur (11). Development may proceed further in indeterminate nodules, and low rates of nitrogen fixation can sometimes be detected (3, 5). Two forms of LPS exist: LPS I, consisting of lipid A, core tri-and tetrasaccharides, and the O-chain polysaccharide, and LPS II, which lacks the O-chain polysaccharide (10, 16). R. leguminosarum bv. viciae RU301 has been isolated and characterized as a Tn5 mutant that contains only LPS II (12). Complementing clones were obtained from both R. leguminosarum bv. phaseoli and R. leguminosarum bv. viciae gene libraries, and a common clustering of LPS and dct genes was demonstrated (12).Noel (10) defined five regions involved in LPS biosynthesis in Rhizobium etli CE3: the ␣-, ␤-, and ␥-lps regions, exoBC, and a poorly defined region which contains the lps-166 mutation. Strain RU301 is not complemented by cosmids corresponding to the ␣-, ␤-, or ␥-lps or the exoB region (12). A 3.4-kb EcoRI-HindIII fragment from the cosmid pIJ1848 (9), which is derived from R. leguminosarum bv. phaseoli, complemented strain RU301 (Fig. 1) and was used as a probe to determine if there was any homology to cosmid DNA encoding the ␣-, ␤-, or ␥-lps region. Hybridization analysis supported the view that the complementing region constituted a region separate from those previously described (12). The 3.4-kb fragment also complements the mutant strain VF-39-86 (14), which has been characterized as having a truncated core tetrasaccharide component containing the disaccharide mannose (1-5) 3-deoxy-Dmanno-2-octulosonic acid (Kdo) (16). A smaller PstI-EcoRI subclone, pRU74 (2.4 kb), complemented strain RU301 but not VF-39-86, indicating that at least two genes involved in LPS core tetrasaccharide synthesis may be present in this previously undefined region (12).Subclones derived from pRU74 were cycle sequenced [ Exo FIG. 1. Map of the LPS region of R. leguminosarum bv. phaseoli. pIJ1848 complements the LPS mutant strains RU301 and VF-39-86. The position of Tn...

Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

“…lpcA was partially sequenced and was proposed to encode the galactosyltransferase because of its homology to other sugar transferases and chemical characterization of LPS isolated from an lpcA::Tn5 insertion mutant (24 -26). lpcB was sequenced entirely, and although it showed no homology to any known gene, it was proposed to encode the distal Kdo-transferase based upon the absence of the distal Kdo in the LPS core isolated from an lpcB transposon insertion mutant (24). We now demonstrate by means of our enzyme assays that lpcA does indeed encode the galactosyltransferase and that lpcB encodes the distal Kdo-transferase.…”

“…X94963. The full-length lpcA gene shows moderate homology to many bacterial glycosyltransferase genes, including some of those involved in the assembly of LPS cores (2,4,24). For instance, the lpcA gene displays 30%, 27%, and 26% identity, respectively, to lgtC of Neisseria gonorrhoeae (45), to ipa-12d of Bacillus subtilis (46), and to rfaJ (waaJ) (2, 4) of E. coli.…”

“…We now describe the three structural genes of R. leguminosarum that encode these glycosyltransferases. Two of the genes, lpcA and lpcB, adjacent to each other on the chromosome, were reported previously (24,25), based on mutants with truncated core LPS structures. lpcA was partially sequenced and was proposed to encode the galactosyltransferase because of its homology to other sugar transferases and chemical characterization of LPS isolated from an lpcA::Tn5 insertion mutant (24 -26).…”

Glucomannan-Mediated Attachment ofRhizobium leguminosarumto Pea Root Hairs Is Required for Competitive Nodule Infection

“…viciae 3841. Several genes for production of extracellular EPSs have been identified in different strains and biovars of R. leguminosarum, but the only polysaccharide mutants described so far for the sequenced strain 3841 are mutants with mutations that affect lipopolysaccharide biosynthesis (1,34). To identify EPS genes in strain 3841, we performed BLAST searches (3) using protein sequences from known polysaccharide biosynthetic genes from different rhizobia.…”

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

“…Previous chemical/genetic studies (48) indicated that QuiNAc was linked to a Kdo residue (Fig. 1), and biosynthetic/ genetic studies (44,45,49,50) provide strong evidence that this Kdo is part of the biosynthetic core of the LPS. It follows from our composition data and these previous conclusions that QuiNAc not only is part of the O antigen but is the first sugar added in O-antigen synthesis.…”