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• W1964552051 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. 1H-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. leguminosarum- legume symbiosis. 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. 1H-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. leguminosarum- legume symbiosis. Rhizobium, Bradyrhizobium, and Azorhizobium are bacterial genera that form N2-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(1Peters N.K. Frost J.W. Long S.R. Science. 1986; 233: 977-980Crossref PubMed Scopus (493) Google Scholar, 2Redmond J. Batley M. Djordjevic M. Innes R. Kuempel P. Rolfe B. Nature. 1986; 323: 632-635Crossref Scopus (328) Google Scholar, 3Lerouge P. Roche P. Faucher C. Maillet F. Truchet G. Promé J.C. Dénarié J. Nature. 1990; 344: 781-784Crossref PubMed Scopus (818) Google Scholar, 4Spaink H.P. Sheeley D.M. van Brussel A.A.N. Glushka J. York W.S. Tak T. Geiger O. Kennedy E.P. Reinhold V.N. Lugtenberg B.J.J. Nature. 1991; 354: 125-130Crossref PubMed Scopus (400) Google Scholar). These Nod factors are chitolipooligosaccharides (CLOSs) 1The abbreviations used are: CLOSchitolipooligosaccharidekbkilobase pair(s)FID-GCflame ionization detection gas chromatographyESI-MSelectrospray ionization mass spectrometryFAB-MSfast atom bombardment mass spectrometryGC/MSgas chromatography/mass spectrometrynodnodulationpSymsymbiotic plasmidHPLChigh performance liquid chromatography. consisting of β-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.O-acetyl, sulfate, etc.) that make their biological activity host-specific(5Carlson R.W. Price N.P.J. Stacey G. Mol. Plant-Microbe Interact. 1994; 7: 684-695Crossref PubMed Scopus (107) Google Scholar). 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(6Dénarié J. Debellé F. Rosenberg C. Annu. Rev. Microbiol. 1992; 46: 497-531Crossref PubMed Scopus (234) Google Scholar, 7Hirsch A.M. New Phytol. 1992; 122: 211-237Crossref PubMed Scopus (471) Google Scholar). chitolipooligosaccharide kilobase pair(s) flame ionization detection gas chromatography electrospray ionization mass spectrometry fast atom bombardment mass spectrometry gas chromatography/mass spectrometry nodulation symbiotic plasmid high performance liquid chromatography. 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 host-specific (nodFERL, nodMN) nod genes residing within a 14-kb region on its resident symbiotic plasmid (pSym)(8Schofield P. Ridge R. Rolfe B. Shine J. Watson J. Plant Mol. Biol. 1984; 3: 3-11Crossref PubMed Scopus (55) Google Scholar, 9Djordjevic M.A. Weinman J.J. Aust. J. Plant Physiol. 1991; 18: 543-557Google Scholar). 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(10Djordjevic M. Schofield P. Rolfe B. Mol. & Gen. Genet. 1985; 200: 463-471Crossref Scopus (108) Google Scholar, 11Spaink H.P. Weinman J. Djordjevic M.A. Wijffelman C.A. Okker R.J.H. Lugtenberg B.J.J. EMBO J. 1989; 8: 2811-2818Crossref PubMed Scopus (60) Google Scholar). Tn5 disruption of nodE (but not genes downstream of nodE) in ANU843 results in a unique dual phenotype, which is defective in nodulation of white clover, and gain in the ability to nodulate a new host, peas(10Djordjevic M. Schofield P. Rolfe B. Mol. & Gen. Genet. 1985; 200: 463-471Crossref Scopus (108) Google Scholar). Evaluations of the nodE sequence and of CLOS structures made by certain recombinant nod-overexpressing strains have led to the current model proposing that NodE is a 3-ketoacyl synthase that controls the host range of R. leguminosarum bv. viciae and bv. trifolii by specifying the synthesis of unique conjugated tri- and tetraunsaturated fatty acid moieties with characteristic absorption maxima between 300 and 330 nm in CLOS species(4Spaink H.P. Sheeley D.M. van Brussel A.A.N. Glushka J. York W.S. Tak T. Geiger O. Kennedy E.P. Reinhold V.N. Lugtenberg B.J.J. Nature. 1991; 354: 125-130Crossref PubMed Scopus (400) Google Scholar, 12Spaink H.P. Bloemberg G.V. van Brussel A.A.N. Lugtenberg B.J.J. van der Drift K.M.G.M. Haverkamp J. Thomas-Oates J.E. Mol. Plant-Microbe Interact. 1995; 8: 155-164Crossref Scopus (56) Google Scholar). It was originally thought that Rhizobium synthesizes only minute quantities of CLOSs and excretes them into the extracellular milieu where they can act on the host plant(3Lerouge P. Roche P. Faucher C. Maillet F. Truchet G. Promé J.C. Dénarié J. Nature. 1990; 344: 781-784Crossref PubMed Scopus (818) Google Scholar, 4Spaink H.P. Sheeley D.M. van Brussel A.A.N. Glushka J. York W.S. Tak T. Geiger O. Kennedy E.P. Reinhold V.N. Lugtenberg B.J.J. Nature. 1991; 354: 125-130Crossref PubMed Scopus (400) Google Scholar, 13Price N.P.J. Relic B. Talmont F. Lewin A. Promé D. Pueppke S.G. Maillet F. Dénarié J. Promé J.C. Broughton W.J. Mol. Microbiol. 1992; 6: 3575-3584Crossref PubMed Scopus (164) Google Scholar). However, we recently found that CLOS glycolipids can be purified in significantly higher yield (>1000-fold) from extracts of cell membranes of wild-type rhizobia than from culture supernatants(14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar, 15Cedergren R. Ross K. Hollingsworth R.I. Biochemistry. 1995; 34: 4467-4477Crossref PubMed Scopus (35) Google Scholar). In the present study, we have critically evaluated the proposed function of nodE by performing detailed structural analyses of CLOS species made by wild-type ANU843, an isogenic nodE::Tn5 mutant derivative ANU297, and a recombinant strain ANU845 pRtRF101 containing the cloned 14-kb HindIII pSym nod region of ANU843 on multiple copy plasmid pWB5a introduced into the pSym-cured derivative ANU845. Our experiments reveal that ANU843 makes a large diversity of major and minor CLOS species, which does not change with impairment of nodE function or increased nod copy number. This spectrum of CLOSs does not, however, include molecules containing tri- or tetraunsaturated fatty acids. This necessitated a reevaluation of results of a recent report (12Spaink H.P. Bloemberg G.V. van Brussel A.A.N. Lugtenberg B.J.J. van der Drift K.M.G.M. Haverkamp J. Thomas-Oates J.E. Mol. Plant-Microbe Interact. 1995; 8: 155-164Crossref Scopus (56) Google Scholar) in which six CLOS species from R. trifolii were proposed to contain nodE-dependent tri- or tetraunsaturated fatty acids. We show here that the mass spectral ions to which these latter structures were assigned are attributable to sodium adducts of the major nodE-independent CLOS species. Portions of this work were presented recently. 2S. Philip-Hollingsworth, G. Orgambide, J. Bradford, J. Lee, D. Smith, R. Hollingsworth, and F. Dazzo, poster presented at the 10th International Congress on Nitrogen Fixation, May 28 to June 3, 1995, St. Petersburg, Russia. R. trifolii wild-type strain ANU843, its isogenic nodE::Tn5 mutant derivative ANU297, and its pSym-cured derivative ANU845 were obtained from B. Rolfe, Australian National University(10Djordjevic M. Schofield P. Rolfe B. Mol. & Gen. Genet. 1985; 200: 463-471Crossref Scopus (108) Google Scholar, 16Rolfe B. Gresshoff P. Shine J. Plant Sci. Lett. 1982; 19: 277-284Crossref Google Scholar). ANU843 has only one copy of nodE and produces a flavonoid-inducible protein detected by immunoblotting with polyclonal anti-NodE protein antiserum, whereas no flavonoid-inducible protein reactive with the same antiserum is detected in ANU297(10Djordjevic M. Schofield P. Rolfe B. Mol. & Gen. Genet. 1985; 200: 463-471Crossref Scopus (108) Google Scholar, 11Spaink H.P. Weinman J. Djordjevic M.A. Wijffelman C.A. Okker R.J.H. Lugtenberg B.J.J. EMBO J. 1989; 8: 2811-2818Crossref PubMed Scopus (60) Google Scholar). Plasmid pRtRF101 contains the 14-kb HindIII pSym nod region of ANU843 (8Schofield P. Ridge R. Rolfe B. Shine J. Watson J. Plant Mol. Biol. 1984; 3: 3-11Crossref PubMed Scopus (55) Google Scholar) cloned on pWB5a(17Fisher R.F. Tu J.K. Long S.R. Appl. Environ. Microbiol. 1985; 49: 1432-1435Crossref PubMed Google Scholar), a derivative of the IncP-1 plasmid pRK290 that is maintained in five to eight copies/genome equivalent(18Ditta G. Stanfield S. Corbin D. Helinsky D.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7347-7351Crossref PubMed Scopus (1890) Google Scholar). Strain ANU845 pRtRF101 (from A. Squartini, Padova University) was constructed by triparental conjugation between the Escherichia coli donor strain HB101 pRtRF101 (from S. Long, Stanford University,(17Fisher R.F. Tu J.K. Long S.R. Appl. Environ. Microbiol. 1985; 49: 1432-1435Crossref PubMed Google Scholar)) and the pSym-cured recipient ANU845(16Rolfe B. Gresshoff P. Shine J. Plant Sci. Lett. 1982; 19: 277-284Crossref Google Scholar), using as a helper plasmid pRK2013 hosted in E. coli JM109. E. coli strains were grown at 37°C in TY medium containing 10 μg/ml tetracycline for the donor and 30 μg/ml kanamycin for the helper. Rhizobium recipients were grown in TY medium at 28°C. 10 ml of mid-exponential phase cultures of each strain were pelleted. Cells were resuspended in 100 μl of saline solution, and 50-μl aliquots were mixed on nitrocellulose 0.22-μm filters on TY plates and incubated for 24 h. Cells on filters were resuspended in 2 ml of saline solution, and aliquots were plated on defined BIII medium (19Dazzo F.B. Burns R.G. Slater J.H. Experimental Microbial Ecology. Blackwell Scientific Publications, Oxford, United Kingdom1982: 431-446Google Scholar) containing 2 μg/ml tetracycline. Transconjugants were purified by restreaking, and their identity was confirmed by plasmid profile analysis using a modified Eckhardt gel technique(20Espuny M.R. Ollero F.J. Bellogin R.A. Ruiz-Sainz J.E. Perez-Silva J. J. Appl. Bacteriol. 1987; 63: 13-20Crossref Scopus (35) Google Scholar). Axenic seedlings of Dutch white clover (Trifolium repens L.) were grown on slopes of nitrogen-free Fahraeus agar as described previously(19Dazzo F.B. Burns R.G. Slater J.H. Experimental Microbial Ecology. Blackwell Scientific Publications, Oxford, United Kingdom1982: 431-446Google Scholar). Axenic seedlings of pea (Pisum sativum cv. Alcan) were grown in Erlenmeyer flasks containing 200 ml of Fahraeus agar(21Djordjevic M.A. Zurkowski W. Rolfe B.G. J. Bacteriol. 1982; 151: 560-568Crossref PubMed Google Scholar). Inocula were grown at 30°C for 5 days on BIII agar (plus 30 μg/ml kanamycin for ANU297 and 2 μg/ml tetracycline for ANU845 pRtRF101). For nodulation tests, cells were diluted in Fahraeus medium and applied at a dose of 5 × 107 cells/plant, with 12 plant replicates. Plants were incubated in a plant growth chamber (22Salzwedel J.L. Dazzo F.B. Mol. Plant-Microbe Interact. 1993; 6: 127-134Crossref PubMed Scopus (35) Google Scholar) and examined periodically for emergence of root nodules with confirmation of their structure after root clearing(23Truchet G. Camut S. de Billy F. Odorico R. Vasse J. Protoplasma. 1989; 149: 82-88Crossref Scopus (73) Google Scholar). Uninoculated controls received sterile Fahraeus medium only. Cells were grown with constant shaking (150 rpm) at 30°C in 2-liter flasks containing 1 liter of BIII broth with 0.45 mM CaCl2(24McKay I.A. Djordjevic M.A. Appl. Environ. Microbiol. 1993; 59: 3385-3392Crossref PubMed Google Scholar) and 4 μM 4′,7-dihydroxyflavone(2Redmond J. Batley M. Djordjevic M. Innes R. Kuempel P. Rolfe B. Nature. 1986; 323: 632-635Crossref Scopus (328) Google Scholar). BIII medium was further supplemented with 30 μg/ml kanamycin or 2 μg/ml tetracycline for ANU297 and ANU845 pRtRF101, respectively. Batch cultures were grown to post-exponential phase (9 × 108 cells/ml) just preceding stationary phase (25Dazzo F.B. Urbano M.R. Brill W.J. Curr. Microbiol. 1979; 2: 15-20Crossref Scopus (26) Google Scholar, 26Hrabak E.M. Urbano M.R. Dazzo F.B. J. Bacteriol. 1981; 148: 697-711Crossref PubMed Google Scholar), harvested by centrifugation at 10,000 × g for 45 min at 4°C, and further washed with deionized water. CLOSs were extracted from cells from 4 liters of broth culture using chloroform/1-propanol/methanol/water (1:2:2:3) and then fractionated by a series of reversed-phase chromatographies (14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar) to yield the isolated family of CLOSs. 500 MHz 1H-NMR spectroscopy (14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar) was performed on 6-mg samples at 25°C in deuterated dimethyl sulfoxide with suppression of the water resonance. The chemical shifts were measured relative to an external trimethylsilane standard. UV absorption spectra were recorded in ethanol on a Varian DMS200 spectrophotometer. For compositional analyses, 3-mg samples were treated with 1 ml of 2% HCl in methanol at 80°C for 16 h and then evaporated to dryness and partitioned between 2 ml of water/chloroform (1:1). The fatty acid methyl esters extracted into the chloroform layer were identified by FID-GC and GC/MS analyses with comparison to authentic standards whenever possible. The aqueous layer containing the methyl glycosides was evaporated to dryness, peracetylated with acetic anhydride/pyridine (1:1) at 25°C for 18 h, and analyzed by GC/MS. FID-GC analyses of the fatty acid methyl esters were performed on a Varian 3740 gas chromatograph using a J& Scientific DB-1 column (program: 150-300°C at 3°C/min with a 10-min hold at 300°C). Fatty acid methyl esters and peracetylated methyl glycosides were analyzed on a Hewlett-Packard 5995C GC/MS instrument using a J& Scientific DB225 column (program: 150-230°C at 3°C/min with a 20-min hold at 230°C). ESI-MS analyses were performed on a Fisons Platform instrument using an electrospray inlet in either negative or positive ion mode, a mobile phase of acetonitrile/water (1:1), and a flow rate of approximately 10 μl/min. The instrument was tuned for unit resolution, and the capillary voltage was set at approximately 3 kV. The supernatants from 2 liters of broth cultures of ANU843 and ANU297 were passed through C18 reverse phase silica beds (150 ml) followed by washing with 500 ml of water and then with 500 ml of 100% acetonitrile to release adsorbed lipophilic material. The acetonitrile eluate was concentrated to dryness and analyzed by HPLC using a Vydac C18 reverse phase column (linear gradient of 30-100% acetonitrile in water over 50 min, 1 ml/min, monitoring of the effluent at 303 nm). Methyl ester derivatives were prepared from equivalent cell pellets (27Osterhout G.J. Shull V.H. Dick J.D. J. Clin. Microbiol. 1991; 29: 1822-1830Crossref PubMed Google Scholar) and analyzed by GC/MS. CLOS samples were dissolved in 20% acetonitrile in water and injected onto a Vydac C18 reverse phase analytical HPLC column (linear gradient of 20-100% acetonitrile in water over 60 min, 20-min hold at 100% acetonitrile, 0.6 ml/min). The effluent was monitored by a Waters 990 photodiode array detector, with acquisition of the full range UV spectrum of each 303-nm absorbing eluted peak. Wild-type ANU843 efficiently nodulated all of the white clover plants but neither nodulated nor induced cortical cell divisions on pea roots. In contrast, ANU297 took 6 days longer to induce the first emerging nodules on white clover and incited 80% fewer nodules/plant by 1 month. ANU297 reisolated from surface-sterilized nodules of white clover retained the Kanr marker. In addition, ANU297 induced foci of cortical cell divisions resembling root nodule primordia and/or root nodules on 83% of the pea plants. These results were consistent with previously reported symbiotic phenotypes for these strains(10Djordjevic M. Schofield P. Rolfe B. Mol. & Gen. Genet. 1985; 200: 463-471Crossref Scopus (108) Google Scholar). In comparison with ANU843 on white clover, the recombinant strain ANU845 pRtRF101 was twice as efficient in early nodulation kinetics and induced 50% more nodules/plant by 1 month (figure not shown). 3Figures not shown can be provided upon request to the authors. The final yields of membrane CLOSs from pelleted cells of ANU297 (~2-3 mg/liter) and ANU845 pRtRF101 (~19 mg/liter) were 5-fold lower and slightly higher, respectively, than the previously reported yield from an equivalent culture of ANU843 grown identically(14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar). Apart from resonances due to residual traces of 1-propanol, the 1H-NMR spectra of CLOSs from ANU843 and ANU297 were the same (Fig. 1). These spectra contained resonances of methylene and methyl protons from the fatty acyl chain (1.2 and 0.8 ppm, respectively), N-acetyl and O-acetyl groups (1.8-2.0 ppm), the methylene group adjacent to vinyl and carbonyl groups (2.0 and 2.1 ppm, respectively), the carbohydrate ring protons (3.0-4.5 ppm), and the vinyl protons of an isolated double bond (5.3 ppm). A set of common downfield signals between 7.0 and 7.8 ppm were attributed to a combination of amide and conjugated vinyl protons since the former protons exchanged upon treatment with deuterium oxide, whereas the latter remained intact (Fig. 1, insets). GC/MS analyses of the peracetylated methyl glycosides indicated that glucosamine was the sole glycosyl component of CLOSs from ANU843 and ANU297 (figures not shown). The FID-GLC and GC/MS profiles of the fatty acid methyl esters from these same samples were also identical in every detail (Fig. 2). The major fatty acid component was cis-vaccenic acid (C18:1) with lesser amounts of C18:0, C16:0, and C16:1 fatty acids, consistent with previous results(14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar). Together these accounted for approximately 95% of the total fatty acids in the family of CLOSs from both strains. Additional fatty acids detected in both samples by FID-GC and GC/MS were C18:2 and C20:1. Selected ion chromatograms for the characteristic fragment at m/z 103 (28Hollingsworth R.I. Dazzo F.B. J. Microbiol. Methods. 1988; 7: 295-302Crossref Scopus (3) Google Scholar) also revealed 3-hydroxy-C14:0, 3-hydroxy-C16:0, and 3-hydroxy-C18:0 fatty acids (Fig. 2, C and D). Thus, nine different fatty acids are definitively identified in the family of CLOSs from ANU843 and ANU297. Heterogeneity of CLOS species from ANU843 and ANU297 was further analyzed by both negative and positive mode ESI-MS. Like all soft ionization methods, ESI-MS is quite sensitive to the exact purity of the sample (concentrations of metal ions, anions, etc.) over which very little control can be exerted. Although the masses and relative abundance of ions are reproducible in multiple analyses of the same sample, the absolute values of ion intensities from different preparations usually vary to some extent and therefore should not be used in quantitation. Evaluation of high mass ions in several runs of positive and negative mode ESI-MS analyses showed that ANU843 produced a very diverse family of CLOSs (Fig. 3, A and C), and this same diversity was detected in CLOSs from ANU297 (Fig. 3, B and D), in agreement with the above 1H-NMR and GC/MS analyses. This diversity of CLOS species found by these methods consists of non-O-acetylated, mono-O-acetylated, and di-O-acetylated chitotri-, tetra-, and pentasaccharides bearing a large variety of amide-linked fatty acids (Table 1). This list of CLOS species from ANU843 includes the diversity of a previously published list (14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar) plus the di-O-acetylated CLOS species, III(C18:1, 2Ac) with M- at m/z 933, IV(C16:0, 2Ac) with M- at m/z 1110, and IV(C18:1, 2Ac) with M- at m/z 1136 found by negative mode ESI-MS (Table 1).Tabled 1 Open table in a new tab The occurrence of the six high mass ions previously assigned to unique nodE-dependent CLOS species of R. trifolii(12Spaink H.P. Bloemberg G.V. van Brussel A.A.N. Lugtenberg B.J.J. van der Drift K.M.G.M. Haverkamp J. Thomas-Oates J.E. Mol. Plant-Microbe Interact. 1995; 8: 155-164Crossref Scopus (56) Google Scholar) was further investigated by positive mode ESI-MS. All CLOS species from ANU843 and ANU297 identified in negative mode ESI-MS spectra were also found in positive mode ESI-MS spectra (Fig. 3, C and D), either as pseudomolecular ions [M+H]+ (i.e. M+1 u) or as their sodium adducts [M+Na]+ (i.e. M+23 u). An important feature of the positive mode ESI-MS spectra of isolated CLOSs from ANU843 and ANU297 was the occurrence of major additional ions at m/z 1075, 1091, 1117, 1119, 1320, 1322 (Fig. 3, C and D). These ions were assigned to sodium adducts of the major CLOS species found in both strains because they are not detected in the negative mode ESI-MS spectra of the same samples, and their masses are fully consistent with the [M+Na]+ pseudomolecular ions of major CLOS species, namely IV(C18:1) at m/z 1075, IV(C16:0,Ac) at m/z 1091, IV(C18:1,Ac) at m/z 1117, IV(C18:0,Ac) at m/z 1119, V(C18:1,Ac) at m/z 1320, and V(C18:0,Ac) at m/z 1322. These same sodium adducts occur in variable intensities in the positive mode FAB mass spectra of these same samples (figures not shown). A second major contribution of negative mode ESI-MS analyses of CLOS was the detection of fragment ions between m/z 220-360, which could be assigned to various long chain fatty acylamino groups. Each of the fatty acids identified above by GC/MS was also found by negative mode ESI-MS (Fig. 4, A and B). In addition, the ESI-MS analyses in the low mass range revealed several other ions tentatively assigned to saturated, unsaturated, and hydroxylated fatty acids ranging in chain length from C14 to C22 (Fig. 4, A and B). Once again, no qualitative differences were found in this diverse family of definitively and tentatively identified CLOS fatty acids from ANU843 and ANU297 (Table 2).Tabled 1 Open table in a new tab The possibility that nodE-dependent fatty acids containing conjugated polyunsaturations might be present in ANU843 CLOS was further addressed by UV absorption spectroscopy, since three or four double bonds in conjugation with the fatty acid carbonyl group would have characteristic absorption maxima with very high molar extinction coefficients (****, 50,000-100,000) in the region of 300-330 nm(29Williams D.H. Fleming I. Spectroscopic Methods in Organic Chemistry. McGraw-Hill Publishing Co. Ltd., London1966: 6-39Google Scholar). A comparison of the UV absorption spectra of the CLOSs from ANU843 and ANU297 showed no differences in the region of 300-330 nm, indicating that they had the same degree and types of unsaturation (Fig. 5, A and B). The remote possibility that polyunsaturated nodE-dependent CLOSs might selectively accumulate in the extracellular milieu rather than in cell membranes was explored by reverse phase HPLC of the hydrophobic components isolated from the culture supernatants. These analyses indicated that every 303-nm adsorbing peak in the profile of the ANU843 supernatant extract was also found in the profile of the ANU297 supernatant (figures not shown). No 303-nm absorbing peaks eluted between 20-35 min, the region where CLOSs typically elute under these chromatographic conditions. We also evaluated the possibility that unique nodE-dependent fatty acids might have been excluded from our protocol to purify membrane CLOSs. GC/MS analyses detected no differences in total cellular fatty acids from ANU843 and ANU297 grown identically to express their nod genes (Fig. 6, A and B). Some of these cellular fatty acids are found in both CLOSs and phospholipids of ANU843 (C16:0, C16:1, C18:0, C18:1, C18:2, and C20:1), some in both CLOSs and lipopolysaccharide (3OH-C14:0, 3OH-C16:0 and 3OH-C18:0), and others only in phospholipids (C14:0, C17:0Δ, and C19:0Δ)(14Orgambide G.G. Lee J. Hollingsworth R.I. Dazzo F.B. Biochemistry. 1995; 34: 3832-3840Crossref PubMed Scopus (40) Google Scholar, 30Hollingsworth R.I. Carlson R.W. J. Biol. Chem. 1989; 264: 9300-9303Abstract Full Text PDF PubMed Google Scholar, 31Orgambide G.G. Huang Z.-H. Gage D.A. Dazzo F.B. Lipids. 1993; 28: 975-979Crossref PubMed Scopus (20) Google Scholar). Other minor peaks were not characterized since they were found in samples from both ANU843 and ANU297 (Fig. 6, insets). Next, we tested the possibility that production of nodE-dependent CLOSs from R. trifolii might be increased to detectable levels by using recombinant strain ANU845 pRtRF101 containing a higher gene dosage of the entire 14-kb pSym nod region from ANU843 in its pSym-cured background. Positive mode ESI-MS analysis of membrane CLOSs from this strain (Fig. 3E) revealed the same spectrum of CLOS species represented by their pseudomolecular ions [M+H]+ and/or sodium adducts [M+Na]+ as found in ANU843 (Fig. 3C) and ANU297 (Fig. 3D) (see Table 1for ion assignments). Most importantly, no new molecular species of CLOS were found in this recombinant strain. Negative mode ESI-MS of the CLOS sample from the recombinant strain also did not reveal any new species in the high mass range (figure not shown). Furthermore, a careful examination of the fatty acyl amino ions in the low mass region of this negative mode ESI-MS spectrum indicated that its diversity of CLOS-associated fatty acids was identical to both ANU843 and ANU297 (compare Fig. 4C to Fig. 4, A and B). Finally, we investigated the chemical nature of the 303 nm-absorbing components present in membrane CLOS fractions from ANU843, ANU297, and ANU845 pRtRF101 using reverse-phase HPLC with photodiode array detection (Fig. 7). The proportion of these components in the various samples was insignificant (less than one ten-thousandth of a percent of the total mixture), since the peaks were nearly nonexistent despite extremely high molar extinction coefficients (ɛ, 50,000-100,000) for tri- and tetraunsaturated conjugated esters(29Williams D.H. Fleming I. Spectroscopic Methods in Organic Chemistry. McGraw-Hill Publishing Co. Ltd., London1966: 6-39Google Scholar). Even if CLOS species containing such fatty acids constituted 1% of the total mixture, the peaks corresponding to them on the HPLC profile would be at least 1000-fold higher than the regular saturated and monounsaturated fatty acylated species. The converse was observed. The" @default.
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• W1964552051 cites W1513827803 @default.
• W1964552051 cites W1514450428 @default.
• W1964552051 cites W1587051062 @default.
• W1964552051 cites W1608894477 @default.
• W1964552051 cites W1722399083 @default.
• W1964552051 cites W1847993210 @default.
• W1964552051 cites W1973121898 @default.
• W1964552051 cites W1976944441 @default.
• W1964552051 cites W1978399367 @default.
• W1964552051 cites W1984081877 @default.
• W1964552051 cites W1987355768 @default.
• W1964552051 cites W2002214299 @default.
• W1964552051 cites W2010387651 @default.
• W1964552051 cites W2011273061 @default.
• W1964552051 cites W2012318736 @default.
• W1964552051 cites W2012958685 @default.
• W1964552051 cites W2014442268 @default.
• W1964552051 cites W2043637247 @default.
• W1964552051 cites W2048115265 @default.
• W1964552051 cites W2049261586 @default.
• W1964552051 cites W2065396611 @default.
• W1964552051 cites W2067032775 @default.
• W1964552051 cites W2076248870 @default.
• W1964552051 cites W2086101621 @default.
• W1964552051 cites W2088088526 @default.
• W1964552051 cites W2093988186 @default.
• W1964552051 cites W2110129108 @default.
• W1964552051 cites W2119014281 @default.
• W1964552051 cites W2143114242 @default.
• W1964552051 cites W2171800753 @default.
• W1964552051 cites W2315638629 @default.
• W1964552051 cites W2320516078 @default.
• W1964552051 cites W2325700189 @default.
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