Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis Received for publication,March 10, 2020, andin revised form, June 11, 2020 Published, Papersin Press, June 16, 2020, DOI 10.1074/jbc.RA120.013393 Flaviana Di Lorenzo 1 ,Immacolata Speciale 1 ,Alba Silipo 1 ,Cynthia Alías-Villegas 2 ,Sebastián Acosta-Jurado 2 , Miguel-Ángel Rodríguez-Carvajal 3 ,Marta S. Dardanelli 4 ,Angelo Palmigiano 5 ,Domenico Garozzo 5 , José-Enrique Ruiz-Sainz 2 ,Antonio Molinaro 1, *, and José-María Vinardell 2, * From the 1 Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, 2 Department of Microbiology, Faculty of Biology, University of Seville, Sevilla, Spain, 3 Deparment of Organic Chemistry, Faculty of Chemistry, University of Seville, Sevilla, Spain, 4 Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto-INBIAS, CONICET, Córdoba, Argentina, and 5 Istituto per i Polimeri, Compositi e Biomateriali IPCB, Consiglio Nazionale delle Ricerche, Catania, Italy Edited by Chris Whitfield Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glyc- ero-L-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing b -configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its a -configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues.Notably,thisrkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum. Rhizobia are soil bacteria able to establish a symbiotic association with legumes in which a complex interchange of molecular signals between the prokaryotic and the eukaryotic partners takes place. This interchange of molecular signals culminates in the formation of specialized plant structures, called nodules, on the roots or stems of the plant host (1). The molecular dialogue between the bacteria and the plant is initiated by flavonoids exuded by the legume root, which activate the transcription of bacterial nodulation genes. Some of these genes encode proteins involved in the biosynthesis and secretion of molecular signals called Nod factors. These bacterial signals mediate species specificity and induce the development of root nodules in the plant (1–3). In addition to Nod factors, different rhizobial surface polysaccharides (RSP) are commonly necessary for successful nodulation. Exopolysaccharides (EPS), lipopolysaccharides (LPS), capsular polysaccharides (KPS or K-antigen polysaccharides), and cyclic glucans (CG) are the main rhizobial polysaccharides involved in symbiosis. For each specific pair of rhizobium-leg- ume symbionts, one or more RSP could act as signal molecules promoting bacterial infection and/or preventing plant defense reactions (4–7). Sinorhizobium fredii HH103 (8) is a fast-growing rhizobial strain that nodulates Glycine max (soybean) and many other determinate- and indeterminate-nodule forming legumes (9). This bacterium produces at least five different RSP: EPS, LPS, CG, and two types of KPS. The chemical structure of these RSP, except for the LPS, have been previously described (10– 13). Interestingly, two KPS are produced by S. fredii HH103, one is a homopolysaccharide built up of a pseudaminic acid derivative, i.e. a -configured 5-acetamido-3,5,7,9-tetradeoxy-7- (3-hydroxybutyramido)-L-glycero-L-manno-nonulosonic acid [ a -Pse5NAc7(3OHBu)] (12), whereas the other is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) (13). To our knowledge, S. fredii HH103 mutants affected in the production of the poly-Kdo KPS have not been described. An S. fredii Rif R exoA mutant, unable to produce EPS, did not show any detectable reduction in the symbiotic capacity with soybean (10). On the contrary, soybean plants inoculated with HH103 mutants unable to produce CG only formed pseudonodules that did not fix nitrogen (11). Interestingly, S. fredii HH103 Rif R mutants in genes located in the so-called rkp-1 region (such as HH103 rkpA) showed a severe reduction of nodulation with soybeans (14–16). HH103 rkpA failed to produce the poly- a -Pse5NAc7(3OHBu) KPS, but the other RSP did not show any detectable change. Even more importantly, S. fredii HH103 Rif R mutants in genes located in the so-called rkp-3 region (such as rkpM,rkpP,orrkpQ) did not produce the a -Pse5NAc7 (3OHBu) KPS as well, showed altered LPS electrophoretic This article contains supporting information. *For correspondence: José-María Vinardell, [email protected]; Antonio Molinaro, [email protected]. J. Biol. Chem. (2020) 295(32) 10969–10987 10969 © 2020 Di Lorenzo et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. ARTICLE This is an Open Access article under the CC BY license. profiles, and only induced the formation of soybean pseudonodules unable to fix nitrogen (17). LPS are the predominant constituents in the external leaflet of Gram-negative outer membrane and can be conceptually divided in three different structural regions: a glycolipid component called lipid A that is linked to a core oligosaccharide (core OS) that is attached to an outer polysaccharide, termed the O- chain (18). On the basis of the nature of the saccharide portion, an LPS can be classified as smooth, semirough, or rough: smooth-type LPS (S-LPS) contains all three abovementioned domains, whereas a semirough-type LPS (sR-LPS) has a subunit (complete or truncated) of the O-chain, and a rough-type LPS (R-LPS) does not express any O-chain moiety (18). To the best of our knowledge, the complete LPS structure has only been described for five different rhizobial strains: Rhizobium etli CE3, R. leguminosarum biovar viciae 3841, R. leguminosarum biovar viciae 128C53 (19), and Bradyrhizobium sp. strain BTAi1 and ORS285 (20–22); in addition, only partial structures or the chemical composition of different LPS parts of various Sinorhizobium strains have been reported (23). Nevertheless, it is widely known that rhizobial LPSs appear to play very relevant roles at different stages of the symbiotic interaction with legumes, including recognition and infection, invasion of root cortical cells, bacterial penetration into nodule cells, and formation and persistence of functional symbiosomes (1,19,24–31). Indeed, it has been demonstrated that alterations in the LPS structure can potently affect the symbiotic properties of the bacterium; in particular, O-chain rhizobial mutants were reported to be drastically affected in symbiosis, either in the infection event, nodule development, or bacteroid differentiation, and this phenomenon has been observed with both determinate and indeterminate nodule-forming legumes (32–36). In this work, we focused attention on the LPS isolated from two S. fredii HH103 Rif R mutants: HH103 rkpA (16)and HH103 rkpM (17). HH103 rkpA was chosen because it does not produce KPS, which could interfere with LPS purification, and its LPS silver- and immune-staining profiles were equal to those of S. fredii HH103 Rif R , indicating that the LPS structure of both strains should be highly similar or identical. In parallel, HH103 rkpM was investigated because it does not produce KPS as well, and its LPS silver-staining profile was shown to be only slightly altered with respect to S. fredii HH103 Rif R . Structural investigation showed that HH103 rkpA possesses an LPS containing b -Pse5NAc7(3OHBu), which was never described before as a component of this polysaccharide in rhizobia. Interestingly, we also show that S. fredii HH103 rkpM produces an LPS devoid of such pseudaminic acid derivatives, and that this mutant is severely impaired in symbiosis with the HH103 host legume Macroptilium atropurpureum. Results Silver staining and antibody recognition of the LPS produced by different S. fredii HH103 mutants affected in LPS and/or KPS biosynthesis In previous works (17,31,37), we have studied different S. fredii HH103 mutants (in the rkpM,lpsB,lpsE,rkpK,andlpsL genes) that were affected in LPS production, as revealed by alterations in their LPS electrophoretic profiles. Relevant characteristics of these mutants are summarized in Table 1. The lpsB and lpsE genes belong to the greA lpsB lpsCDE cluster and are predicted to code for type 1 glycosyltransferases that transfer activated sugars to a variety of substrates, including glycogen and LPS (31). The rkpK and lpsL genes constitute the socalled rkp-2 region, and their encoded products are responsible for the synthesis of glucuronic and galacturonic acid, respectively (37). Finally, the rkpM gene belongs to the rkpLMNOPQ operon (rkp-3 region), which is involved in the production of the pseudaminic acid derivative that constitutes the HH103 KPS (17). In this work, we have compared their LPS profiles in a single polyacrylamide gel (Fig. 1A) to classify them according to the mobility of the silver-stained LPS bands. We hypothesized that any increase of the electrophoretic mobility of the LPS bands would be directly related to the size of the truncated LPS forms produced by each mutant. Therefore, mutants showing faster migrating LPS bands might correspond to mutants producing smaller truncated LPS core OS forms. WT S. fredii HH103 Rif R and its rkpA mutant derivative were also included as controls, because this mutant does not produce KPS but its LPS shows the same electrophoretic profile of HH103 Rif R (16). Figure 1Aallowed us to compare the electrophoretic alterations exhibited by the different mutants. As shown in this figure, the LPS profile of S. fredii HH103 Rif R is composed of four silver-stained bands of slower mobility and two thick bands of faster mobility. This observation suggested that bands in the upper part of the gel would correspond to LPS molecules made up of lipid A, core OS, and O-chain (or part of it), whereas in the lower part of the gel a smaller LPS, migrating as a dispersed thick region, was assumed to contain the lipid A and core OS with no O-chain subunits. The biological nature of the tiny smears visualized in the upper part of the gel remains unknown. At the bottom of each lane appears a diffuse band that is not always visualized in SDS-PAGE gels carried out with HH103 LPS samples (16,17) and whose nature is unknown. Table 1 Previously studied Sinorhizobium fredii HH103 mutants affected in LPS Mutated gene Predicted function of the encoded product RSP affected a Symbiotic phenotype with soybean Reference lpsB Glycosyl transferase LPS* High reduction in the number of Fix 1 nodules, pseudonodules 31 lpsE Glycosyl transferase LPS* lpsL UDP-glucuronate 4-epimerase LPS* Pseudonodules, Fix – 37 rkpK UDP-glucose 6-dehydrogenase LPS* EPS 2 Nod 1 Fix 1 (as WT) rkpM Pyridoxal phosphate (PLP)-dependent aminotransferase LPS* KPS 2 Pseudonodules, Fix – 17 a LPS* refers to alterations in the electrophoretic profile and lack of recognition by the mAb NB6-228.22. Sinorhizobium fredii HH103 LPS structure and symbiosis 10970 J. Biol. Chem. (2020) 295(32) 10969–10987 As reported before (17,31,37), the electrophoretic mobility of the bands of the five HH103 mutants affected in LPS was clearly faster than that of HH103 Rif R or its rkpA derivative. However, there were also differences among the different LPS mutants (Fig. 1A): mutants in lpsL,rkpK,andlpsE showed similar LPS profiles with faster electrophoretic mobility than that of HH103 rkpM but slower than that of HH103 lpsB. Following these observations, we employed NB6-228.22, a mAb able to recognize all the silver-stained LPS bands of S. fredii HH103 Rif R (38). As shown in Fig. 1B, the pattern of LPS bands recognized by NB6-228.22 in S. fredii HH103 Rif R was equal to that in HH103 rkpA. Neither the tiny smears at the upper part nor the diffuse bands at the bottom of the gels reacted with the antibody. None of the S. fredii HH103 Rif R mutants showing alterations in their LPS electrophoretic profiles (rkpM,lpsL,rkpK,lpsE,andlpsB) were recognized by NB6-228.22, indicating that in all these mutants the LPS epitope recognized by NB6-228.22 was missing or has become unavailable for antibody recognition. Introduction in HH103 rkpM of a cosmid containing the HH103 rkp-3 region (strain HH103 rkpM C) restored the WT LPS profile (Fig. 1A) and recognition by the mAb NB6-228.22 (Fig. 1B). LPS profiles of S. fredii HH103 Rif R free-living cells is undistinguishable from HH103 Rif R bacteroids isolated from G. max or C. cajan (39). However, S. fredii HH103 Rif R bacteroids isolated from Glycyrrhiza uralensis showed altered LPS profiles compared with those of S. fredii HH103 free-living cells (39). Given these premises, the LPS from bacteroids of G. uralensis nodules induced by inoculation with HH103 Rif R or HH103 rkpM were also analyzed by SDS-PAGE in the present study (Fig. 1A).TheLPSprofileofHH103rkpM bacteroids was slightly different from that of free-living HH103 rkpM cells and clearly altered with respect to that of HH103 bacteroids. This demonstrated that HH103 rkpM LPS is altered not only in free-living cells but also in G. uralensis bacteroids compared with those of the WT strain. The LPS of HH103 rkpM bacteroids isolated from Glycyrrhiza nodules was not recognized by the mAb NB6-228.22 (Fig. 1B). Taking into account all the above results, we started the structural characterization of the LPS of HH103 mutants rkpA and rkpM. Structural determination of the LPS from S. fredii HH103 rkpA and HH103 rkpM mutants To define the detailed structure of S. fredii HH103 rkpA and HH103 rkpM LPS, the dried bacterial pellets were extracted by the hot phenol-water procedure (40), checked by SDS-PAGE, and extensively purified. To define the nature of the monosaccharides composing the isolated LPSs, a set of chemical analyses (41) on pure water phase extracts was performed. The merging of data from compositional analysis, through acetylated methyl glycosides derivatization, and absolute configuration, through octyl glycoside derivatization, revealed the presence in both LPS of D-galacturonic (D-GalA) and D-glucuronic Figure 1. Electrophoretic profile and immunodetection of the LPS produced by different S. fredii HH103 Rif R mutants affected in LPS and/or KPS production. A, SDS-PAGE after silver staining of LPS crude extracts. B, immunostaining using the mAb NB6-228.22 of LPS crude extracts. In both panels A and B, the mutated gene is indicated on the top of the lane. HH103 denotes HH103 Rif R , and rkpM C denotes the complemented rkpM mutant. All of the samples were extracted from free-living cells, with the only exception being samples HH103 b and rkpM b, which correspond to samples extracted from bacteroids from Glycyrrhiza uralensis nodules induced by HH103 Rif R and its rkpM mutant, respectively. Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10971 (D-GlcA) acids, D-glucose (D-Glc), D-galactose (D-Gal), 2- amino-2-deoxy-D-glucose (D-GlcN), and Kdo. Linkage analyses (41) proved the pyranose form for all sugar residues and allowed the identification of the same branching points for the monosaccharides composing both the LPS, except for terminal-D-GalpA, which was found only in the case of HH103 rkpM, and 2-substituted-D-GalpA, which was found instead only in the case of HH103 rkpA; on the other hand, 4-substi- tuted D-GalpA, terminal and 2-substituted D-GlcpA, 2-substi- tuted and 3-substituted D-Glcp, 3,6-disubstituted D-Galp,6- substituted-D-GlcpN, and terminal and 4,5-disubstituted Kdo were detected in both LPS. Importantly, as described further in the text, an additional sugar constituent, Pse5NAc7(3OHBu) (Pse), was identified solely in HH103 rkpA and only after an indepth investigation of the isolated LPS saccharide component using NMR spectroscopy and MALDI-TOF MS. To define the primary structure of the saccharide portion of both HH103 rkpA and HH103 rkpM LPS, an aliquot of each sample underwent a mild acid hydrolysis to selectively cleave the linkage between the Kdo, which is the first sugar component of the core OS, and the lipid A moiety. After several steps of purification comprising size-exclusion low-pressure and HPLC, the pure saccharide moieties (OS1 rkpA and OS1 rkpM ) were analyzed via 1D and 2D NMR spectroscopy, including double-quantum filtered phase-sensitive correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), rotating frame Overhauser enhancement spectroscopy (ROESY), heteronuclear single quantum coherence ( 1 H- 13 C HSQC), 1 H- 13 C HSQC-TOCSY, and heteronuclear multiple bound correlation ( 1 H- 13 C HMBC). All sugar units were present as pyranose rings according to the 13 C chemical shift values and in full accordance with chemical analyses. The analysis of both the 1 H NMR (Fig. 2)and HSQC spectra (Fig. 3) of the pure OS1 rkpA clearly showed the occurrence of eight anomeric signals attributed to eight different spin systems (A–H,Table 2), whereas the presence of a Kdo unit was attested by the presence of the diasterotopic H-3 methylene proton signals at d H 1.94/1.75 ppm (K, d C 33.9 ppm, Fig. 2 and 3). Furthermore, the 1 H NMR spectrum (Fig. 2) showed the presence of four intense methyl signals at d H 1.05, 1.09, 1.11, and 1.13 ppm (Table 2) and of another intense methyl signal at 1.90 ppm accounting for two N-acetyl groups (NAc). Finally, the presence of additional methylene proton signals at 1.55/2.42 ppm (Pse I H-3 ax /H-3 eq , d C 36.0 ppm) and 1.50/2.47 ppm (Pse II H-3 ax /H-3 eq , d C 36.1 ppm) were also identified. Starting from the above methylene signals, the analysis of HSQC and HMBC spectra, supported by the correlations visible in COSY, TOCSY, and ROESY spectra, allowed the complete structural elucidation of two Pse residues (here indicated as Pse I and Pse II ). Briefly, the diasterotopic methylene protons H-3 Pse I showed long-range correlations with an oxymethine carbon atom (C-4 Pse I , d C 66.3 ppm), a quaternary carbon (C-2 Pse I , d C 103.2 ppm), and a carboxylic group (C-1 Pse I , d C 172.8 ppm) (Fig. 4). A less intense HMBC correlation between H-3 ax Pse I and a nitrogen-bearing carbon atom resonating at 48.0 ppm (C-5 Pse I ) was also detected (Fig. 4) and identified as the site of N-acetylation, as proven by the NOE correlation between H-5 Pse I (4.05 ppm) and the methyl proton at 1.90 ppm (not shown). Starting from H-5 Pse I , a long-range correlation with an oxymethine carbon atom at 73.0 ppm, assigned as C-6 Pse I , was visible in the HMBC spectrum (Fig. 4); further on, the methyl proton signal at 1.09 ppm correlated with an oxymethine carbon atom (C-8 Pse I , 68.3 ppm) and a nitrogenbearing carbon (C-7 Pse I , 53.7 ppm), with the latter, in turn, correlated to H-6 Pse I (3.77 ppm) (Fig. 4). Finally, a 3-hydroxy- butyryl moiety (HBu, made up of the CH 3 -C group at d C 22.0 ppm, the C-CHOH-C group at d C 69.8 ppm, the C-CH 2 -C at d C 45.0 ppm, and the CO group at 173.4 ppm) was found to be connected by amide linkage at position 7 of Pse I ; the location of the HBu moiety was attained by the observation of a longrange correlation between C-19(173.4 ppm) and H-7 Pse I (3.94 ppm) in the HMBC spectrum (Fig. 4). Because of the relatively large J H-3ax,H-4 (;12.9 Hz) and the small J H-4,H-5 and J H-5,H-6 (,5 Hz) coupling constants, it was possible to deduce the axial orientation of H-4 Pse I and the equatorial orientation of H-5 Pse I that combined with the J H-6,H-7 coupling constant of ;10 Hz, suggested the L-glycero-L-manno configuration of unit Pse I (42). This was also corroborated by the observation of the chemical shift for the C-9 signal at 16.2 ppm (Table 2), which is typical of the L-epimer of the glycero-L-manno isomers; in contrast, chemical shifts at around 20 ppm are observed in the case of the D-epimers (42). Interestingly, the large difference between the H-3 ax and H-3 eq chemical shifts (0.88 ppm) indicated the axial orientation of the carboxyl group, which is the b configuration of Pse I at the anomeric center (42). Finally, the downfield displacement of the chemical shift of C-39(69.8 ppm) of unit Pse I suggested a site of glycosylation at such a position. Similarly, a second unit of b -Pse5NAc7(3OHBu) has been assigned (Pse II ), but it turned out to be a terminal residue, linked to position 39of Pse I , as proven by the long-range correlation between the C-2 of Pse II and the oxymethine proton H- 39(4.13 ppm, Table 2 and Fig. 4)ofPse I . The absolute configuration of the HBu substituent remains to be defined. As for the other spin systems, A(H-1 at 5.53 ppm, Table 2), B(H-1 at 5.50 ppm), C(H-1 at 5.09 ppm), F(H-1 at 4.55 ppm), and H(H-1 at 4.23 ppm) were identified as hexuronic acid residues because of the correlation of their H-5 signals with carboxyl groups resonating at 175.3, 175.1, 175.5, 174.9, and 174.2 ppm, respectively. The 3 J H-3,H-4 and 3 J H-4,H-5 coupling constant values (3 and 1 Hz, respectively) measured for A,B, and Fwere diagnostic of a galacto configuration, whereas the large ring 3 J H, H coupling constants observed for Cand Hled to their identification as gluco-configured sugar units. The a -anomeric configuration for A,B, and Cwas attributed on the basis of the intraresidual NOE contact of H-1 with H-2 and the 3 J H1,H2 coupling constant values, whereas the b -anomeric configuration for residues Fand Hwas inferred by the large 3 J H-1,H-2 values and the NOE contacts of H-1 with H-3 and H-5. Therefore, Aand B were identified as a -GalA units, Cas an a -GlcA, Fas a b -GalA, and Has a b -GlcA. Residue D(H-1 at 5.04 ppm, Table 2)was identified as a -Gal because of the small 3 J H,H values for H-3/H- 4 and H-4/H-5, whereas residues E(H-1 at 5.00 ppm) and G (H-1 at 4.26 ppm) were assigned to a -and b -Glc, respectively. Low-field shifted carbon signals (Table 2 and Fig. 3) were useful to identify substitution at O-4 of residues Aand B,atO- 2ofFand H, at O-3 and O-6 of D,atO-3ofG, at O-4 and O-5 Sinorhizobium fredii HH103 LPS structure and symbiosis 10972 J. Biol. Chem. (2020) 295(32) 10969–10987 of K, and, as described above, at O-39of Pse I ; on the other hand, residues C,E,andPse II were ascribed to terminal sugar units. The sequence of the monosaccharide residues was deduced by using both NOE correlations attained from the ROESY spectrum and the long-range correlations of the HMBC spectrum. In detail, the scalar interresidue long-range connections H-1 D/C-5 K, H-1 E/C-4 K,H-1 C/C-3 D, H-1 G/ C-6 D, H-1 A/C-3 G, H-1 H/C-4 A, H-1 B/C-2 H, H-1 F/C-4 B, H-2 F/C-2 Pse I ,and H-39Pse I /C-2 Pse II (Fig. 4) were identified and led to the structural assessment of the OS1 rkpA product (Fig. 5). To gain further structural information about HH103 rkpA LPS, an aliquot of sample underwent a complete deacylation step by hydrazinolysis followed by strong alkaline treatment. This approach provides the complete oligosaccharide moiety comprising the disaccharide lipid A backbone and allows us to evaluate the presence of substituents that could have been lost during the mild acid hydrolysis procedure. Indeed, despite the great heterogeneity observed in the product because of the harsh alkaline treatment and the intrinsic nature of the oligosaccharide, the NMR analysis of the obtained product (OS2 rkpA ) clearly disclosed, in addition to the signals relative to the a -GlcN (X)and b -GlcN (Y) of the lipid A (Table 2,Fig. S1), the occurrence of a further Kdo unit (J) whose methylene proton signals resonated at 1.86/2.11 ppm ( d C 33.9 ppm). Finally, the observation of the low-field position of the signal for C-2 (75.4 ppm) of the a -Glc (E 2 ) linked to the 4,5- a -Kdo (K 2 ), and the occurrence of an NOE correlation between H-3 Jand H-2 of E 2 led to the determination of the sequence J-(2!2)-E 2 - (1!4)-K 2 , with the latter Kdo unit as the one linked to b -GlcN (Y) of lipid A. Therefore, summarizing all results arising from mild acid hydrolysis and full deacylation procedures, we found that HH103 rkpA LPS possesses a novel carbohydrate backbone made up of a disaccharide of b -Pse5NAc7(3OHBu), two Kdo units, one a -D-Glcp, one D- b -Glcp,oneD- a -Galp, two D- a -GalpA, one D- b -GalpA, one D- a -GlcpA and one D- b -GlcpA, as sketched in Fig. 5. A similar approach was used to elucidate the structure of OS1 rkpM (Table S1). The comparison of the NMR spectra of OS1 rkpM and OS1 rkpA (overlapped HSQC spectra are reported in Fig. S2) revealed an identical structure apart from the absence of any Pse residue in OS1 rkpM . Indeed, as shown by both the 1 H and the HSQC NMR spectra (Fig. 2 and Fig. S2), no signals relative to the methylene and methyl groups of Pse residues were visible. Consequently, the b -GalA unit F' of OS1 rkpM (Table S1), corresponding to residue Fin the rkpA mutant, was identified as a terminal sugar because of the lack of glycosylation at position O-2 by the Pse moiety. Therefore, the structure of the saccharide backbone of rpkM mutant LPS is as reported in Fig. 5. In the case of HH103 rkpM, the NMR investigation (not shown) of the fully deacylated product (OS2 rkpM ) confirmed the occurrence of a terminal Kdo unit, lost during the mild acid hydrolysis treatment, as also proven by MALDI-TOF MS analysis executed on intact HH103 rkpM LPS (see below). Figure 2. 1 H NMR spectra of Sinorhizobium fredii HH103 rkpA (top)andrkpM (bottom). Anomeric signals of spin systems are designated as described in Table 2. Diastereotopic methylene proton signals of Pse I ,Pse II , and Kdo residues (K/K') are also indicated. Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10973 MALDI-TOF MS of intact LPS from HH103 rkpA and rkpM mutants An aliquot of pure LPS isolated from HH103 rkpA and HH103 rkpM has been investigated by MALDI-TOF MS. The MS spectra, recorded in negative polarity, are reported in Fig. 6. The linear MALDI-TOF MS spectrum of HH103 rkpA LPS (Fig. 6A) showed at higher molecular masses (between m/z 3500 and 4800) a series of [M–H] – ions relative to the native LPS mixture, composed of species differing by the composition of both the acyl and the sugar moieties. More importantly, ion fragments arising from the cleavage of the labile glycosidic bond between Kdo and the lipid A portion were also identified at lower mass ranges (m/z1500–2100). This LPS fragmentation ( b -elimination) yielding both oligosaccharide ions and lipid A ions furnished important details about the structure of HH103 rkpA LPS. In particular, the MALDI-TOF MS investigation gave us the chance to appreciate the heterogeneity of both the lipid A and the core OS portion of such LPS. Indeed, the main ion fragments in Fig. 6Aat m/z1770.2 corresponded to an OS composed of four hexuronic acids (HexA), three hexoses (Hex), one Kdo, and one Pse (OS A ), namely, an OS species matching with the one identified in the NMR analysis but lacking one Pse and one HexA unit. More interestingly, the ion fragment at m/z 2165.7 was attributed to an OS made up of five HexA, three Hex, two Kdo, and one Pse (OS B ), confirming the occurrence of a second unit of Kdo, as observed in the NMR investigation of the fully deacylated product. Finally, the main LPS species, as stated above, were consistent with a combination of the OS ion peaks and differently acylated lipid A species. Similarly, the reflectron MALDI-TOF MS spectrum of intact LPS from the HH103 rkpM mutant revealed the occurrence of a main fragment ion at m/z1409.5 relative to an OS built up of four HexA, three Hex, and one Kdo (OS A ); OS fragments composed by a further Kdo unit (m/z1629.7, OS B ) and HexA (m/z 1805.5, OS C ) have also been identified. Therefore, by MALDITOF MS analysis it was possible to further confirm that HH103 rkpM expresses an LPS devoid of Pse, according to NMR data. As described for the rkpA mutant, in the higher mass region, LPS species composed of the above OS species and differently acylated lipid A forms have been detected. Fatty acid compositional analysis and MALDI-TOF MS of lipid A from HH103 rkpM and rkpA LPS Fatty acid compositional analysis revealed the occurrence, in both HH103 mutants, of 12:0(3-OH), 13:0(3-OH), 14:0(3-OH), 15:0(3-OH), 16:0(3-OH), 17:0(3-OH), 18:0(3-OH), 19:0(3-OH), 18:1(3-OH), 18:2(3-OH), 19:1(3-OH), 20:1(3-OH), and the very-long-chain fatty acids (VLCFA) 28:0(27-OH), 30:0(29- OH), 28:0(27-OHBu), and 30:0(29-OHBu), which is consistent with the lipid A fatty acid composition from the genus Figure 3. Zoom of the 1 H- 13 C HSQC spectrum of OS1 rkpA product obtained after mild acid hydrolysis of the LPS from S. fredii HH103 rkpA.Most of the heteronuclear correlations are indicated. Spin system labels are indicated as described in Table 2. The color of the cross-peaks indicates the phase: black indicates CH and CH 3 signals, whereas red indicates CH 2 signals. Sinorhizobium fredii HH103 LPS structure and symbiosis 10974 J. Biol. Chem. (2020) 295(32) 10969–10987 Sinorhizobium (23). To unveil the structure of the glycolipid portion of the LPS from both mutants, an aliquot of the mild acid hydrolysis-precipitated product, containing lipid A, has been analyzed by negative-ion MALDI-TOF MS (the spectrum is reported in Fig. S3, Aand B). The MALDI mass spectra of the isolated lipid A fractions were identical for both mutants and further confirmed, and expanded on, the high heterogeneity of such LPS moieties and the occurrence of hydroxybutyrate as an acyloxyacyl substituent carried on some of the VLCFAs. The main ion peaks and the proposed interpretation of the substituting fatty acids on the lipid A backbone are reported in Table S2. As an example, in the higher mass regions, the ion peak at m/z2066.0 was attributed to a bis-phosphorylated penta-acy- lated lipid A species carrying two 14:0(3-OH), one 18:0(3-OH), and one 19:0(3-OH) as primary fatty acids and one 30:0(29- OHBu) as the secondary acyl moiety; further on, the ion peak at m/z1952.0 corresponded to a penta-acylated lipid A species decorated by two phosphates and carrying two 14:0(3-OH), one 18:0(3-OH), one 19:0(3-OH), and one 28:0(27-OH), whose relative species missing one 14:0(3-OH) was assigned to a peak at m/z1725.9. At m/z2050.0, a further penta-acylated lipid A form decorated by two phosphates, two 14:0(3-OH), one 18:0 (3-OH), one 18:1(3-OH), and one 30:0(29-OHBu) was also detected (Fig. S3C); the related species carrying only one phosphate group and lacking one 14:0(3-OH) moiety was identified at m/z1743.9. The more intense ion peak at m/z1870.0 was assigned to bis-phosphorylated lipid A forms composed of two 14:0(3-OH), one 13:0(3-OH), one 12:0(3-OH), and one 28:0(27- OHBu), whose relative species lacking one 14:0(3-OH) was identified at m/z1643.9. The mAb NB6-228.22 recognizes S. fredii HH103 Rif R LPS but fails in the recognition of KPS Structural investigation demonstrated that a b -configured Pse5NAc7(3OHBu) is present in the LPS of rkpA but absent from rkpM. Because NB6-228.22 binds to the LPS produced by rkpA but fails in the recognition of rkpM LPS, we investigated whether this mAb was also able to recognize the KPS produced by HH103 Rif R , which is a homopolymer of a -Pse5NAc7 (3OHBu) (12). For this purpose, intact cells of S. fredii HH103 Rif R and its derivatives mutated in the rkpA,rkpM,rkpK,lpsL,lpsB,and lpsE genes were transferred to a nylon membrane and were deposited on a positively charged nylon transfer membrane and immunostained with NB6-228.22. In this experiment, purified LPS and KPS samples from HH103 were also analyzed. As shown in Fig. 7A, only cells from the two strains not affected in Table 2 Proton and carbon chemical shifts of the OS1 rkpA (A-H, K, Pse I and Pse II ) and OS2 rkpA (E 2 ,K 2 , J, X, and Y) products derived from mild acid hydrolysis and complete deacylation, respectively, of LPS from S. fredii HH103 rkpA Product Chemical shift Value at position a : 1/192/293/394/4956789 A 1 H 5.53 4.03 3.69 4.28 4.77 4- a -D-GalA 13 C 99.4 68.3 71.0 82.5 71.1 175.3 B 1 H 5.50 3.99 3.68 4.27 4.81 4- a -D-GalA 13 C 99.5 68.4 71.0 81.6 71.0 175.1 C 1 H 5.09 3.68 3.88 4.19 4.49 t- a -D-GlcA 13 C 101.2 68.7 69.5 70.5 71.9 175.5 D 1 H 5.04 3.53 3.79 4.19 3.91 4.09/3.99 3,6- a -D-Gal 13 C 99.7 71.6 82.3 69.5 70.4 66.7 E 1 H 5.00 3.45 3.75 3.38 3.95 3.84/3.64 t- a -D-Glc 13 C 97.4 70.3 70.5 70.3 72.9 60.4 F 1 H 4.55 3.92 3.88 4.20 4.07 2- b -D-GalA 13 C 99.9 76.2 74.0 72.1 75.4 175.4 G 1 H 4.26 3.18 3.62 3.38 3.19 3.52 3- b -D-Glc 13 C 103.5 70.7 81.1 68.5 76.2 62.2 H 1 H 4.23 3.44 3.40 3.31 3.42 2- b -D-GlcA 13 C 102.6 81.3 76.1 68.9 76.0 174.2 K 1 H 1.94/1.75 4.03 3.95 3.69 3.87 3.67/3.49 4,5- a -D-Kdo 13 C ND 96.2 33.9 65.5 75.0 70.9 69.7 63.0 Pse I1 H 2.42/1.55 3.68 4.05 3.77 3.94 4.01 1.09 39- b -L,L-Pse 13 C 172.8 103.2 36.0 66.3 48.0 73.0 53.7 68.3 16.2 HBu 1 H 2.24 4.13 1.13 13 C 173.4 45.0 69.8 22.0 Pse II 1 H 2.47/1.50 3.77 4.05 3.65 3.86 3.99 1.05 tb -L,L-Pse 13 C 172.1 102.3 36.1 66.6 48.0 73.9 53.5 68.8 16.9 HBu 1 H 2.22 4.06 1.11 13 C 173.3 44.9 64.8 21.9 NAc 1.90 21.8 X 1 H 5.55 3.30 3.80 3.47 4.06 4.16/3.68 6- a -D-GlcN 13 C 90.4 54.1 69.5 70.1 72.4 69.2 Y 1 H 4.78 2.95 3.71 3.40 3.67 3.44 6- b -D-GlcN 13 C 99.3 55.5 71.9 69.6 75.0 61.0 E 21 H 5.08 3.68 3.68 3.26 4.06 3.43 2- a -D-Glc 13 C 97.3 75.4 71.9 69.8 72.0 61.0 K 21 H 1.74/1.99 4.05 4.20 3.62 3.92 3.82/3.55 4,5- a -D-Kdo 13 C ND ND 34.3 70.4 72.2 72.2 70.3 62.8 J 1 H 1.86/2.11 3.83 3.91 3.93 3.87 3.82/3.49 t- a -D-Kdo 13 C ND ND 33.9 69.4 70.1 71.4 69.6 63.0 a 19,29,39, and 49indicate the proton and the carbon positions of the HBu group. The proton chemical shift of the NH of the NAc group for both Pse I and Pse II was at 7.51 ppm, whereas that at position 7 of both Pse I and Pse II was at 7.18 ppm. Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10975 Figure 4. Superimposition of 1 H- 13 C HSQC (blue/red) with 1 H- 13 C HMBC (black)ofOS1 rkpA product. Densities are labeled with the same letters listed in Table 2. The most relevant interresidue long-range correlations involving sugar moieties are indicated in red, whereas in black are reported some important intraresidue long-range correlations. Figure 5. Structural assessments of the OS1 rkpA , OS2 rkpA , OS1 rkpM, and OS2 rkpM products. The complete structure of the core OS from S. fredii HH103 rkpA and rkpM was achieved by combination of data attained from the NMR investigation of the LPS after mild acid hydrolysis (yielding OS1 rkpA and OS1 rkpM ) and full deacylation (yielding OS2 rkpA and OS2 rkpM ) procedures. The mild acid hydrolysis selectively cleaved the linkage between the Kdo and the nonreducing glucosamine of the lipid A portion, enabling the isolation of the core OS moieties (sugar units labeled A,A'-H,H',K,K',Pse I ,andPse II ) from the lipid A domain. The complete deacylation procedure allowed the isolation of the core OS plus the glucosamine disaccharide backbone (labeled X,X',Y, and Y') of the lipid A. The occurrence of an additional Kdo unit (J,J') was deduced by analysis of the fully deacylated products which, consequently, allowed the identification of the spin system E 2 ,E 2 '(i.e. the a -D-Glcpsubstituted at position 2 by the Kdo unit J,J'). The core OS structures are labeled using letters in Table 2 and Table S2. pindicates the pyranose form of sugar residues, whereas Pindicates the phosphate group. Sinorhizobium fredii HH103 LPS structure and symbiosis 10976 J. Biol. Chem. (2020) 295(32) 10969–10987 their LPS (HH103 and its rkpA derivative) as well as the purified LPS sample showed positive recognition by NB6-228.22. These results clearly indicate that NB6-228.22 does not react with HH103 KPS. The epitope recognized by the mAb NB6-228.22 is not common in S. fredii strains Both the LPS and KPS structures may vary at the strain level (1,19). For this reason, we decided to investigate the reactivity of other S. fredii strains against NB6-228.22. Intact cells of 25 S. fredii strains (including HH103) that have been isolated in previous works from soybean plants growing in soils of different Chinese provinces were included in this study (Table S3,Fig. 7B). Only two of these strains (HH103 and HH102), which were isolated from nodules of soybean plants inoculated with soil from Hubei province (43), were recognized by the antibody. The remaining tested strains, isolated and reported in the period 1982–2003 (44), from soils of Chinese East-Central provinces (Shanghai, Hubei, Henan, and Shandong), as well as from the Xingjian Autonomous Region (Western China), were not recognized by NB6-228.22. S. fredii NGR234 and S. meliloti AK631 were included in this study, because their KPS contain two different derivatives of the pseudaminic acid (45). Intact cells of NGR234 and AK631 were not recognized by NB6- 228.22 (Fig. 7B). Immunostaining of PAGE-separated LPS samples from several S. fredii strains confirmed that NB6-228.22 recognized the different LPS bands from HH102 but failed to recognize LPS from strains HGW35, B33, USDA196, USDA197, USDA205, and USDA208 (Fig. 7C). Because the rkp-3 region is involved in the production of Pse5NAc7(3OHBu) (17), all the S. fredii strains tested with NB6-228.22 were also investigated by PCR for the presence of the rkpL,rkpM,rkpP,andrkpQ genes using genomic DNA (gDNA) as the template (Tables S3 and S4). Strain NGR234 was not included in these experiments because its rkp-3 region has been sequenced (46). Only the gDNA of HH103 and HH102 led to the amplification of the expected fragments for all the genes investigated (Table S3), whereas when gDNA of strain S50 was used, the internal fragments of rkpM and rkpP, but not those of rkpL and rkpQ, could be amplified. All amplified fragments showed an identity of 98–100% with the corresponding rkp gene of S. fredii HH103 Rif R (Table S4). Because S. fredii S50 appeared to have only part of the rkpLMNOPQ operon, we investigated the presence of these genes in sinorhizobial strains for which sequences homologous (.85.0 identity at amino acid level) to RkpM were found in silico. The complete operon was present in S. fredii CCBAU05631, Sinorhizobium sp. strain BJ1, S. meliloti Rm41, S. medicae Str9, and S. americanum CCGM7 (Table S5). In two other strains, S. fredii NGR234 and S. saheli LMG7837, the rkpP genes were missing. S. fredii strain CCBAU45436 lacks rkpO, and the rest of the predicted proteins showed identities lower than 55%. Figure 8 shows the organization of the rkp-3 region in S. fredii strainsaswellas in S. meliloti Rm41. Figure 6. A, negative-ion MALDI mass spectrum of intact LPS from S. fredii HH103 rkpA recorded in linear mode. Both LPS molecular ions and their ion fragments, attributable to the core OS (OS A ,OS B ) and lipid A (LipA) species, are visible and indicated in the spectrum. B, negative-ion MALDI mass spectrum of LPS of S. fredii HH103 mutant rkpM recorded in reflectron mode. The lipid A (LipA) and core OS (OS A ,OS B ,OS C ) species are also indicated in the spectrum. Proposed structural assessment of some LPS-related ions has been reported. Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10977 Bacteria were isolated from surface-sterilized nodules as previously reported (38). Bacteria isolated (70 colonies) from 7 nodules induced for each strain on M. atropurpureum or C. cajan roots showed the expected antibiotic resistance markers: Rif R for HH103, Rif R Gm R for HH103 rkpM and rkpA mutants, and Rif R Gm R Tc R for HH103 rkpM C. Microscopy of nodules Optical microscopy studies were carried out as previously described (66). Briefly, M. atropurpureum plants were inoculated and grown for seven weeks in a plant growth chamber. Small fragments of M. atropurpureum nodules then were immediately fixed in 4% (v/v) glutaraldehyde in 0.1 Mcacodylate buffer, pH 7.2, for 2 h at 4 °C after collection. Samples were washed several times in 0.1 Mcacodylate buffer, pH 7.2, dehydrated in acetone at progressively higher concentrations, and embedded in Spurr resine (low-viscosity embedding kit, by Dr. Spurr). The semithin sections (1 m m) were obtained on a Leica EM UC7 ultramicrotome, stained with Toluidine blue, and viewed in an Olympus BX61 light microscope. Extraction of bacteroid cells from nodules Bacteroids from G. uralensis nodules were extracted as previously described (67). Nodules were surface sterilized for 1 min in 100% bleach and then washed three times in water. Nodules were crushed in MMS [40 mM3-(4-morpholino)-propane sulfonic acid, 20 mMKOH, 2 mMMgSO 4 ,0.3Msucrose,pH7.0]buffer, and the liquid containing bacteroids was removed with a pipette. Large particles were removed by passing through a 40- m mcell strainer cap (BD Falcon, BD Biosciences, San Jose, CA, USA). Samples were centrifuged once at 70 3gfor 5 min and posteriorly twice at 2200 3gfor 5 min to resuspend the resulting pellet fraction in MMS medium. LPS extraction from isolated bacteroids was carried out by following the same protocol as that used for the extraction of LPS from free-living cultures (38). Data availability All data described are contained within the manuscript. Acknowledgments—We thank the Centro de Investigación, Tecnología e Innovación (CITIUS), of the University of Seville for light microscopy facilities. Author contributions—F. D. L., I. S., A. S., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., A. M., and J.-M. V. conceptualization; F. D. L., I. S., A. S., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., A. M., and J.-M. V. formal analysis; F. D. L., I. S., A. S., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., A. M., and J.-M. V. funding acquisition; F. D. L., I. S., A. S., C. A.-V., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., and J.-M. V. investigation; F. D. L., I. S., A. S., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., A. M., and J.-M. V. writing-original draft; F. D. L., I. S., A. S., S. A.-J., M.-R.-C., M. S. D., A. P., D. G., J.-E. R.-S., A. M., and J.-M. V. writing-review and editing. Funding and additional information—This work was funded by grants P11-CVI-7500 from the Andalusian Government, and BIO2016-78409-R from the Ministry of Economy and Competitiveness (Spanish Government). M. S. D. is a Career Member of CONICET and worked thanks to grant CONICET PIP 112-201501- 00232. Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article. Abbreviations—The abbreviations used are: KPS, K-antigen polysaccharide; RSP, rhizobial surface polysaccharides; EPS, exopolysaccharides; LPS, lipopolysaccharides; CG, cyclic glucans; OS, oligosaccharide; Pse5NAc7(3OHBu), 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybu- tyramido)-l-glycero-l-manno-nonulosonic acid; DQF-COSY, doublequantum filtered phase-sensitive correlation spectroscopy; TOCSY, total correlation spectroscopy; ROESY, rotating frame Overhauser enhancement spectroscopy; 1 H- 13 C HSQC, heteronuclear single quantum coherence; 1 H- 13 CHMBC,heteronuclearmultiple bound correlation; NAc N-acetyl groups; VLCFA, very-long-chain fatty acids; OD 600 , optical density at 600 nm; GLC-MS, gas LC-MS. References 1. López-Baena, F. J., Ruiz-Sainz, J. E., Rodríguez-Carvajal, M. A., and Vinardell, J. M. (2016) Bacterial molecular signals in the Sinorhizobium frediisoybean symbiosis. Int. J. Mol. Sci.17, 755 CrossRef 2. Gage, D. J. (2004) Infection and invasion of roots by symbiotic, nitrogen fixing rhizobia during nodulation of temperate legumes. Mol. Biol. Rev.68, 280–300 CrossRef Medline 3. Jones, K. M., Kobayashi, H., Davies, B. W., Taga, M. E., and Walker, G. C. (2007) How rhizobial symbionts invade plants: the Sinorhizobium-Medi- cago model. Nat. Rev. Microbiol.5, 619–633 CrossRef Medline 4. Krol, E., and Becker, A. (2009) Surface polysaccharides as fitness factors of rhizospheric nitrogen-fixing bacteria. in Bacterial Polysaccharides: Current Innovations and Future Trends, (Ullrich M., ed), pp. 187–211, Caister Academic Press, London 5. Janczarek, M., Rachwaø, K., Marzec, A., Grzadziel, J., and Palusińska- Szysz, M. (2015) Signal molecules and cell-surface components involved in early stages of the legume–rhizobium interactions. Appl. Soil Ecol.85, 94–113 CrossRef 6. Kawaharada, Y., Kelly, S., Nielsen, M. W., Hjuler, C. T., Gysel, K., Muszyński, A., Carlson, R. W., Thygesen, M. B., Sandal, N., Asmussen, M. H., Vinther, M., Andersen, S. U., Krusell, L., Thirup, S., Jensen, K. J., et al. (2015) Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308–312 CrossRef Medline 7. Kawaharada, Y., Nielsen, M. W., Kelly, S., James, E. K., Andersen, K. R., Rasmussen, S. R., Füchtbauer, W., Madsen, L. H., Heckmann, A. B., Radutoiu, S., and Stougaard, J. (2017) Differential regulation of the Epr3 receptor coordinates membrane-restricted rhizobial colonization of root nodule primordia. Nat. Commun.8, 14534 CrossRef Medline 8. Vinardell, J.-M., Acosta-Jurado, S., Zehner, S., Göttfert, M., Becker, A., Baena, I., Blom, J., Crespo-Rivas, J. C., Goesmann, A., Jaenicke, S., Krol, E., McIntosh, M., Margaret, I., Pérez-Montaño, F., Schneiker-Bekel, S., et al. (2015) The Sinorhizobium fredii HH103 genome: a comparative analysis with S. fredii strains differing in their symbiotic behaviour with soybean. Mol. Plant Microbe Interact.28, 811–824 CrossRef Medline 9. Margaret, I., Becker, A., Blom, J., Bonilla, I., Goesmann, A., Göttfert, M., Lloret, J., Mittard-Runte, V., Rückert, C., Ruiz-Sainz, J. E., Vinardell, J. M., and Weidner, S. (2011) Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean. J. Biotechnol.155, 11–19 CrossRef Medline 10. Rodríguez-Navarro, D. N., Rodríguez-Carvajal, M. A., Acosta-Jurado, S., Soto, M. J., Margaret, I., Crespo-Rivas, J. C., Sanjuan, J., Temprano, F., Gil- Serrano, A., Ruiz-Sainz, J. E., and Vinardell, J. M. (2014) Structure and biological roles of Sinorhizobium fredii HH103 exopolysaccharide. PLoS ONE 9, e115391 CrossRef Medline Sinorhizobium fredii HH103 LPS structure and symbiosis 10984 J. Biol. Chem. (2020) 295(32) 10969–10987 11. Crespo-Rivas, J. C., Margaret, I., Hidalgo, A., Buendía-Clavería, A. M., Ollero, F. J., López-Baena, J., Murdoch, P., Rodríguez-Carvajal, M. A., Soria-Díaz, M. E., Reguera, M., Lloret, J., Sumpton, D. P., Mosely, J. A., Thomas-Oates, J. E., van Brussel, A. A. N., et al. (2009) Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate-nodule forming legumes and overproduce an altered EPS. Mol. Plant Microbe Interact.22, 575–588 CrossRef Medline 12. Gil-Serrano, A. M., Rodríguez-Carvajal, M. A., Tejero-Mateo, P., Espartero, J. L., Menéndez, M., Corzo, J., Ruiz-Sainz, J. E., and Buendía-Clavería, A. M. (1999) Structural determination of a 5-acetamido-3,5,7,9-tetra- deoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonusolonic acidcontaining homopolysaccharide isolated from Sinorhizobium fredii HH103. Biochem. J.342, 527–535 CrossRef 13. Chataigné, G., Couderc, F., and Poinsot, V. (2008) Polysaccharides analysis of sinorhizobial capside by on-line anion exchange chromatography with pulse amperiometric detection and mass spectrometry coupling. J. Chromatogr. A 1185, 241–250 CrossRef Medline 14. Parada, M., Vinardell, J. M., Ollero,F. J., Hidalgo, A.,Gutiérrez, R., Buendía Clavería, A., Lei, W., Margaret, I., López-Baena, F. J., Gil-Serrano, A., Rodríguez Carvajal, M. A., Moreno, J., and Ruiz-Sainz, J. E. (2006) Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajans.Mol. Plant Microbe Interact.19, 43–52 CrossRef Medline 15. Hidalgo, A., Margaret, I., Crespo-Rivas, J. C., Parada, M., Murdoch, P. S., López, A., Buendía-Clavería, A. M., Moreno, J., Albareda, M., Gil-Serrano, A. M., Rodríguez-Carvajal, M. A., Palacios, J. M., Ruiz-Sainz, J. E., and Vinardell, J. M. (2010) The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea. Microbiology 156, 3398–3411 CrossRef Medline 16. Margaret-Oliver, I., Lei, W., Parada, M., Rodríguez-Carvajal, M. A., Crespo-Rivas, J. C., Hidalgo, Á., Gil-Serrano, A., Moreno, J., Rodríguez- Navarro, D. N., Buendía-Clavería, A., Ollero, J., Ruiz-Sainz, J. E., and Vinardell, J. M. (2012) Sinorhizobium fredii HH103 does not strictly require KPS and/or EPS to nodulate Glycyrrhiza uralensis, an indeterminate nodule-forming legume. Arch. Microbiol.194, 87–102 CrossRef Medline 17. Margaret, I., Crespo-Rivas, J. C., Acosta-Jurado, S., Buendía-Clavería, A. M., Cubo, M. T., Gil-Serrano, A., Moreno, J., Murdoch, P. S., Rodríguez-Carvajal, M. A., Rodríguez-Navarro, D. N., Ruiz-Sainz, J. E., Sanjuan, J., Soto, M. J., and Vinardell, J. M. (2012) Sinorhizobium fredii HH103 rkp- 3genes are required for KPS biosynthesis, affect LPS structure and are essential for infection of legumes forming determinate nodules. Mol. Plant Microbe Interact.25, 825–838 CrossRef Medline 18. Di Lorenzo, F., De Castro, C., Lanzetta, R., Parrilli, M., Silipo, A., and Molinaro, A. (2015) Lipopolysaccharides as microbe-associated molecular patterns: a structural perspective. in Carbohydrates in Drug Design and Discovery (Jiménez-Barbero, J., Javier Canada, F., and Martín-Santamaría, S., eds.), pp. 38–63, Royal Society of Chemistry (RSC), London 19. Carlson, R. W., Forsberg, L. S., and Kannenberg, E. L. (2010) Lipopolysaccharides in Rhizobium-legume symbioses. in Endotoxins: Structure, Function and Recognition, (Wang X. and Quinn P. J., eds), pp. 339–386, Springer, New York 20. Silipo, A., Leone, M. R., Erbs, G., Lanzetta, R., Parrilli, M., Chang, W. S., Newman, M. A., and Molinaro, A. (2011) A unique bicyclic monosaccharide from the Bradyrhizobium lipopolysaccharide and its role in the molecular interaction with plants. Angew. Chem. Int. Ed. Engl.50, 12610– 12612 CrossRef Medline 21. Silipo, A., Vitiello, G., Gully, D., Sturiale, L., Chaintreuil, C., Fardoux, J., Gargani, D., Lee, H. I., Kulkarni, G., Busset, N., Marchetti, R., Palmigiano, A., Moll, H., Engel, R., Lanzetta, R., et al. (2014) Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes. Nat. Commun.5, 5106 CrossRef Medline 22. Di Lorenzo, F., Palmigiano, A., Duda, K. A., Pallach, M., Busset, N., Sturiale, L., Giraud, E., Garozzo, D., Molinaro, A., and Silipo, A. (2017) Structure of the lipopolysaccharide from the Bradyrhizobium sp. ORS285 rfaL mutant strain. ChemistryOpen 6, 541–553 CrossRef Medline 23. Gudlavalleti, S. K., and Forsberg, L. S. (2003) Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp. J. Biol. Chem.278, 3957–3968 CrossRef 24. Brewin, N. J., Perotto, S., Kannenberg, E. L., Rae, A. L., Rathburn, E. A., Lucas, M. M., Kardailsky, I., Gunder, A., Bolaños, L., Donovan, N., and Drøbak, B. K. (1993) Mechanisms of cell and tissue invasion by Rhizobium leguminosarum: the role of cell surface interactions. in Advances in Molecular Genetics of Plant-Microbe Interactions (Nester E. W. and Verma D. P. S., eds.), pp. 369–380, Kluwer Academic Publishers, New York 25. Noel, K. D., van den Bosch, K. A., and Kulpaca, B. (1986) Mutation in Rhizobium phaseoli that lead to arrested development of infection threads. J. Bacteriol.168, 1392–1401 CrossRef Medline 26. Stacey, G., So, J. S., Roth, L. E., Lakshmi, B., and Carlson, R. W. (1991) A lipopolysaccharide mutant from Bradyrhizobium japonicum that uncouples plant from bacterial differentiation. Mol. Plant Microbe Interact.4, 332–340 CrossRef Medline 27. Lagares, A., Caetano-Anolles, G., Niehaus, K., Lorenzen, J., Ljunggren, H. D., Pühler, A., and Favelukes, G. (1992) A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa. J. Bacteriol.174, 5941–5952CrossRef Medline 28. Lagares, A., Hozbor, D. F., Niehaus, K., Otero, A. J., Lorenzen, J., Arnold, W., and Pühler, A. (2001) Genetic characterization of a Sinorhizobium meliloti chromosomal region in lipopolysaccharide biosynthesis. J. Bacteriol.183, 1248–1258 CrossRef Medline 29. Campbell, G. R., Reuhs, B. L., and Walker, G. C. (2002) Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core. Proc. Natl. Acad. Sci. U S A 99, 3938–3943 CrossRef Medline 30. Campbell, G. R., Sharypova, L. A., Scheidle, H., Jones, K. M., Niehaus, K., Becker, A., and Walker, G. C. (2003) Striking complexity of lipopolysaccharide defects in a collection of Sinorhizobium meliloti mutants. J. Bacteriol.185, 3853–3862 CrossRef Medline 31. Margaret, I., Lucas, M., Acosta-Jurado, S., Buendía-Clavería, A. M., Fedorova, E., Hidalgo, A., Rodríguez-Carvajal, M. A., Rodríguez-Navarro, D. N., Ruiz-Sainz, J. E., and Vinardell, J. M. (2013) The Sinorhizobium fredii HH103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules. PLoS ONE 8, e74717 CrossRef Medline 32. Carlson, R. W., Kalembasa, S., Turowski, D., Pachori, P., and Noel, K. D. (1987) Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development. J. Bacteriol. 169, 4923–4928 CrossRef Medline 33. Cava, J. R., Elias, P. M., Turowski, D. A., and Noel, K. D. (1989) Rhizobium leguminosarum CFN42 genetic regions encoding lipopolysaccharide structures essential for complete nodule development on bean plants. J. Bacteriol.171, 8–15 CrossRef Medline 34. Noel, K. D., Forsberg, L. S., and Carlson, R. W. (2000) Varying the abundance of O-antigen in Rhizobium etli and its effect on symbiosis with Phaseolus vulgaris.J. Bacteriol.182, 5317–5324 CrossRef Medline 35. Gao, M., D'Haeze, W., De Rycke, R., Wolucka, B., and Holsters, M. (2001) Knockout of an azorhizobial dTDP-L-rhamnose synthase affects lipopolysaccharide and extracellular polysaccharide production and disables symbiosis with Sesbania rostrata.Mol. Plant Microbe Interact.14, 857–866 CrossRef Medline 36. Noh, J. G., Jeon, H. E., So, J. S., and Chang, W. S. (2015) Effects of the Bradyrhizobium japonicum waaL (rfaL) gene on hydrophobicity, motility, stress tolerance, and symbiotic relationship with soybeans. Int. J. Mol. Sci. 16, 16778–16791 CrossRef Medline 37. Acosta-Jurado, S., Navarro-Gómez, P., Crespo-Rivas, J.-C., Medina, C., Murdoch, P. D. S., Cuesta-Berrio, L., Rodríguez-Carvajal, M.-Á., Ruiz- Sainz, J.-E., and Vinardell, J.-M. (2017) The Sinorhizobium fredii HH103 rkp-2 region is involved in the biosynthesis of lipopolysaccharide and exopolysaccharide but not in K-antigen polysaccharide production. Plant Soil 417, 415–431 CrossRef 38. Buendía-Clavería, A. M., Moussaid, A., Ollero, F. J., Vinardell, J. M., Torres, A., Moreno, J., Gil-Serrano, A. M., Rodríguez-Carvajal, M. A., Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10985 Tejero-Mateo, P., Peart, J. L., Brewin, N. J., and Ruiz Sainz, J. E. (2003) A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide. Microbiology 149, 1807–1818 CrossRef Medline 39. Crespo-Rivas, J. C., Guefrachi, I., Mok, K. C., Villaécija-Aguilar, J. A., Acosta-Jurado, S., Pierre, O., Ruiz-Sainz, J. E., Taga, M. E., Mergaert, P., and Vinardell, J. M. (2016) Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the inverted repeat-lacking clade legume Glycyrrhiza uralensis.Environ. Microbiol.18, 2392–2404 CrossRef Medline 40. Westphal, O., and Jann, K. (1965) Bacterial lipopolysaccharides extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem.5, 83–91 41. De Castro, C., Parrilli, M., Holst, O., and Molinaro, A. (2010) Microbeassociated molecular patterns in innate immunity: extraction and chemical analysis of gram-negative bacterial lipopolysaccharides. Methods Enzymol.480, 89–115 CrossRef Medline 42. Knirel, Y. A., Shashkov, A. S., Tsvetkov, Y. E., Jansson, P. E., and Zãhringer, U. (2003) Tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry. Adv. Carbohydr. Chem. Biochem.58, 371– 417 CrossRef 43. Dowdle, S. F., and Bohlool, B. B. (1985) Predominance of fast-growing Rhizobium japonicum in a soybean field in the People’s Republic of China. Appl. Environ. Microbiol.50, 1171–1176 CrossRef 44. Thomas-Oates, J., Bereszczak, J., Edwards, E., Gill, A., Noreen, S., Zhou, J. C., Chen, M. Z., Miao, L. H., Xie, F. L., Yang, J. K., Zhou, Q., Yang, S. S., Li, X. H., Wang, L., Spaink, H. P., et al. (2003) A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China. Syst. Appl. Microbiol.26, 453–465 CrossRef Medline 45. Reuhs, B. L., Geller, D. P., Kim, J. S., Fox, J. E., Kolli, V. S. K., and Pueppke, S. G. (1998) Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens. Appl. Environ. Microbiol.64, 4930–4938 CrossRef Medline 46. Le Quéré, A. J. L., Deakin, W. K., Schmeisser, C., Carlson, R. W., Streit, W. R., Broughton, W. J., and Forsberg, L. S. (2006) Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234. J. Biol. Chem.281, 28981–28992 CrossRef Medline 47. Kiss, E., Kereszt, A., Barta, F., Stephens, S., Reuhs, B. L., Kondorosi, A., and Putnoky, P. (2001) The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure. Mol. Plant Microbe Interact.14, 1395–1403 CrossRef Medline 48. Acosta-Jurado, S., Rodríguez-Navarro, D. N., Kawaharada, Y., Perea, J. F., Gil-Serrano, A., Jin, H., An, Q., Rodríguez-Carvajal, M. A., Andersen, S. U., Sandal, N., Stougaard, J., Vinardell, J. M., and Ruiz-Sainz, J. E. (2016) Sinorhizobium fredii HH103 invades Lotus burttii by crack entry in a Nod factor and surface polysaccharide-dependent manner. Mol. Plant Microbe Interact.29, 925–937 CrossRef Medline 49. Bourassa, D. V., Kannenberg, E. L., Sherrier, D. J., Buhr, R. J., and Carlson, R. W. (2017) The lipopolysaccharide lipid A long-chain fatty acid is important for the Rhizobium leguminosarum growth and stress adaptation in free-living and nodule environments. Mol. Plant-Microbe Interact.30, 161–175 CrossRef Medline 50. Di Lorenzo, F., Sturiale, L., Palmigiano, A., Lembo-Fazio, L., Paciello, I., Coutinho, C. P., Sá-Correia, I., Bernardini, M., Lanzetta, R., Garozzo, D., Silipo, A., and Molinaro, A.(2013) Chemistryand biology of the potent endotoxin from a Burkholderia dolosa clinical isolate from a cystic fibrosis patient. Chembiochem 14, 1105–1115 CrossRef Medline 51. Leontein, K., Lindberg, B., and Lōnngren, J. (1978) Assignment of absolute configuration of sugars by glc of their acetylated glycosides formed from chiral alcohols. Methods Carbohydr. Chem.62, 359–362 CrossRef 52. Holst, O. (2000) Deacylation of lipopolysaccharides and isolation of oligosaccharide phosphates. Methods Mol. Biol.145, 345–353 CrossRef Medline 53. Kasimova, A. A., Shneider, M. M., Arbatsky, N. P., Popova, A. V., Shashkov, A. S., Miroshnikov, K. A., Balaji, V., Biswas, I., and Knirel, Y. A. (2017) Structure and gene cluster of the K93 capsular polysaccharide of Acinetobacter baumannii B11911 containing 5-N-acetyl-7-N-[(R)-3-hydroxybu- tanoyl]pseudaminic acid. Biochemistry 82, 483–489 CrossRef Medline 54. Piantini, U., Sorensen, O. W., and Ernst, R. R. (1982) Multiple quantum filters for elucidating NMR coupling networks. J. Am. Chem. Soc.104, 6800–6801 CrossRef 55. Rance, M., Sørensen, O. W., Bodenhausen, G., Wagner, G., Ernst, R. R., and Wüthrich, K. (2012) Improved spectral resolution in COSY (1)H NMR spectra of proteins via double quantum filtering. Biophys. Res. Commun.425, 527–533 CrossRef 56. States, D. J., Haberkorn, R. A., and Ruben, D. J. (1982) A two-dimensional nuclear Overhauser experiment with pure absorption phase in four quadrants. J. Magnetic Res.48, 286–292 CrossRef 57. Stern, A. S., Li, K. B., and Hoch, J. C. (2002) Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear-prediction extrapolation and maximum-entropy reconstruction. J. Am. Chem. Soc. 124, 1982–1993 CrossRef Medline 58. Sturiale, L., Palmigiano, A., Silipo, A., Knirel, Y. A., Anisimov, A. P., Lanzetta, R., Parrilli, M., Molinaro, A., and Garozzo, D. (2011) Reflectron MALDI TOF and MALDI TOF/TOF mass spectrometry reveal novel structural details of native lipooligosaccharides. J. Mass Spectrom.46, 1135–1142 CrossRef Medline 59. Sturiale, L., Garozzo, D., Silipo, A., Lanzetta, R., Parrilli, M., and Molinaro, A. (2005) New conditions for matrix-assisted laser desorption/ionization mass spectrometry of native bacterial R-type lipopolysaccharides. Rapid Commun. Mass Spectrom.19, 1829–1834 CrossRef Medline 60. Di Lorenzo, F., Palmigiano, A., Al Bitar-Nehme, S., Sturiale, L., Duda, K. A., Gully, D., Lanzetta, R., Giraud, E., Garozzo, D., Bernardini, M. L., Molinaro, A., and Silipo, A. (2017) The lipid A from Rhodopseudomonas palustris strain BisA53 LPS possesses a unique structure and low immunostimulant properties. Chemistry 23, 3637–3647 CrossRef Medline 61. Beringer, J. E. (1974) R factor transfer in Rhizobium leguminosarum.J. Gen. Microbiol.84, 188–198 CrossRef Medline 62. Vincent, J. M. (ed) (1970) The modified Fåhraeus slide technique. in Appendix III, A Manual for the Practical Study of Root Nodule Bacteria,pp 144–145, Blackwell Scientific, New York 63. Robertsen, B. K., Aman, P., Darvill, A. G., McNeil, M., and Albersheim, P. (1981) The structure of acidic extracellular polysaccharides secreted by Rhizobium leguminosarum and Rhizobium trifolii.Plant Physiol.67, 389– 400 CrossRef Medline 64. Vinardell, J. M., Ollero, F. J., Hidalgo, A., López-Baena, F. J., Medina, C., Ivanov-Vangelov, K., Parada, M., Madinabeitia, N., Espuny, M. R., Bellogín, R. A., Camacho, M., Rodríguez-Navarro, D. N., Soria-Díaz, M. E., Gil- Serrano, A. M., and Ruiz-Sainz, J. E. (2004) NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103. Mol. Plant Microbe Interact. 17, 676–685 CrossRef Medline 65. Acosta-Jurado, S., Alias-Villegas, C., Navarro-Gómez, P., Zehner, S., Murdoch, P. D., Rodríguez-Carvajal, M. A., Soto, M. J., Ollero, F. J., Ruiz-Sainz, J. E., Göttfert, M., and Vinardell, J. M. (2016) The Sinorhizobium fredii HH103 MucR1 global regulator is connected with the nod regulon and is required for efficient symbiosis with Lotus burttii and Glycine max cv. Williams. Mol. Plant Microbe Interact.29, 700–712 CrossRef Medline 66. Acosta-Jurado, S., Alias-Villegas, C., Navarro-Gómez, P., Almozara, A., Rodríguez-Carvajal, M. A., Medina, C., and Vinardell, J. M. (2020) Sinorhizobium fredii HH103 syrM inactivation affects the expression of a large number of genes, impairs nodulation with soybean and extends the hostrange to Lotus japonicus.Environ. Microbiol.22, 1104–1124 CrossRef Medline 67. Finan, T. M., McWhinnie, E., Driscoll, B., and Watson, R. J. (1991) Complex symbiotic phenotypes result from gluconeogenic mutations in Rhizobium meliloti.Mol. Plant Microbe Interact.4, 386–389 CrossRef 68. Keyser, H. H., Hu, T. S., Bohlool, B. B., and Weber, D. F. (1982) Fast growing rhizobia isolated from root nodules of soybean. Science 215, 1631– 1632 CrossRef Medline 69. Lamrabet, Y., Bellogín, R. A., Cubo, T., Espuny, M. R., Gil, A., Krishnan, H. B., Megias, M., Ollero, F. J., Pueppke, S., Ruiz-Sainz, J. E., Spaink, H. P., Tejero-Mateo, P., Thomas-Oates, J., and Vinardell, J. M. (1999) Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors Sinorhizobium fredii HH103 LPS structure and symbiosis 10986 J. Biol. Chem. (2020) 295(32) 10969–10987 and significantly decreases competitiveness to nodulate soybeans. Mol. Plant Microbe Interact.12, 207–217 CrossRef Medline 70. Madinabeitia,N.,Bellogín,R.A.,Buendía-Clavería,A.M.,Camacho, M.,Cubo,T.,Espuny,M.R.,Gil-Serrano,A.,deLyra,M.C.C.P.,Moussaid,A.,Ollero,F.J.,SoriaDíaz,M.E.,Vinardell,J.M.,Zeng,J.,andRuiz- Sainz, J. E. (2002) Sinorhizobium fredii HH103 has a truncated nolO gene due to a –1 frameshift mutation that is conserved among other geographically distant S. fredii strains. Mol. Plant Microbe Interact.15, 150–159 CrossRef Medline 71. Trinick, M. J. (1980) Relationships amongst the fast-growing Rhizobium of Lablab purpureus,Leucaena leucocephala,Mimosa sp., Acacia farnesiana, and Sesbania grandiflora and their affinities with other Rhizobium groups. J. Appl. Bacteriol.49, 39–53 CrossRef 72. Casse, F., Boucher, C., Julliot, J. S., Michel, M., and Denarié, J. (1979) Identification and characterization of large plasmids in Rhizobium meliloti using agarose-gel electrophoresis. J. Gen. Microbiol.113, 229–242 CrossRef 73. Kereszt, A., Kiss, E., Reuhs, B. L., Carlson, R. W., Kondorosi, A., and Putnoky, P. (1998) Novel rkp gene clusters of Sinorhizobium meliloti involved in capsular polysaccharide production and invasion of the symbiotic nodule: the rkpK gene codes a UDP-glucose dehydrogenase. J. Bacteriol.180, 5426–5431 CrossRef 74. Perotto, S., Brewin, N. J., and Kannenberg, E. L. (1994) Cytological evidence for a host defense response that reduces cell and tissue invasion in pea nodules by lipopolysaccharide-defective mutants of Rhizobium leguminosarum strain 3841. Mol. Plant Microbe Interact.7, 99–112 CrossRef Sinorhizobium fredii HH103 LPS structure and symbiosis J. Biol. Chem. (2020) 295(32) 10969–10987 10987