Mutation or Increased Copy Number of nodE Has No Effect on the Spectrum of Chitolipooligosaccharide Nod Factors Made By Rhizobiumleguminosarum bv. trifolii

Abstract

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determine unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843.1 H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E. (1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarumlegume symbiosis.Rhizobium, Bradyrhizobium, and Azorhizobium are bacterial genera that form N 2 -fixing nodules on legume roots. In this symbiosis, the plant produces flavonoids that activate bacterial expression of nod genes necessary for production of "Nod factors" involved in infection and nodulation of the corresponding host plant (1-4). These Nod factors are chitolipooligosaccharides (CLOSs) 1 consisting of ␤-1,4-linked oligomers of N-acetylglucosamine bearing an amide-linked fatty acyl moiety at the nonreducing end and may contain other substituents (e.g. Oacetyl, sulfate, etc.) that make their biological activity hostspecific (5). The current model for nod functions is that the common nod genes encode enzymes that synthesize the common backbone of CLOSs, and the host-specific nod genes encode enzymes that introduce these modifications in CLOS structures making them host-specific (6, 7). Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) is the bacterial symbiont of the legume host, clover (Trifolium spp.). In the most thoroughly studied wild-type strain (ANU843), the ability to nodulate white clover is controlled by regulatory (nodD), common (nodABCIJ), and hostspecific (nodFERL, nodMN) nod genes residing within a 14-kb region on its resident symbiotic plasmid (pSym) (8, 9). Elegant studies have shown that NodE is the main determinant of nodulation host range for R. trifolii and its closest relative, the pea symbiont, R. leguminosarum bv. viciae (10, 11). Tn5 disruption of nodE (but not genes downstream of nodE) in ANU843 results in a unique dual phenotype, which is defective in nodula...

Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii

“…The extraction, purification, and structural characterization of membrane CLOSs from ANU843 have been described elsewhere (32,34). A 10 Ϫ5 M stock of the purified CLOS sample (32) was prepared in NF medium, assuming a nominal molecular mass of 1,000 Da.…”

“…However, we recently found that CLOSs accumulate primarily in membranes of wild-type Rhizobium leguminosarum bv. trifolii ANU843 and R. meliloti 2011 (5,32,34). Accumulation of membrane CLOSs in ANU843 requires a functional nodC and nod-activating flavone (32), and these glycolipids are fully capable of eliciting root hair deformation and foci of cortical cell divisions in white clover in the same concentration range as that found for extracellular CLOSs of other rhizobia on their corresponding legume hosts (31).…”

Rhizobium leguminosarum bv. trifolii produces Lipo-chitin Oligosaccharides with nodE-dependent Highly Unsaturated Fatty Acyl Moieties

“…In a recent study, Philip-Hollingsworth et al (8) rejected our assignment of highly unsaturated LCOs and suggested that they have "critically evaluated the proposed function of the nodE gene by performing detailed structural analysis" of LCOs produced by R. leguminosarum bv. trifolii.…”

“…To account for the absence of LCOs with highly unsaturated fatty acyl moieties, Philip-Hollingsworth et al (8) suggest that the ions in the mass spectra assigned as nodE-dependent LCOs by Spaink et al (7) were actually due to sodium adducts of the FIG. 1.…”

New insights into Nod factor biosynthesis: Analyses of chitooligomers and lipo-chitooligomers of Rhizobium sp. IRBG74 mutants

“…The resulting strain secreted amounts of LCOs that were sufficient for analysis. The LCOs secreted into the culture medium by overexpressing strains are not expected to differ significantly from those found in the membranes of wild-type rhizobia [14]. More than 40 different structures were found in the culture supernatant of the nodD -overexpressing strain (Fig.…”