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

JBC Papers in Press. Published on June 16, 2020 as Manuscript RA120.013393

The latest version is at https://www.jbc.org/cgi/doi/10.1074/jbc.RA120.013393

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

symbiosis

Flaviana Di Lorenzo¹, Immacolata Speciale¹, Alba Silipo¹, Cynthia Alías-Villegas², Sebastián

Acosta-Jurado², Miguel-Ángel Rodríguez-Carvajal³, Marta S. Dardanelli⁴, Angelo Palmigiano⁵,

Domenico Garozzo⁵, José-Enrique Ruiz-Sainz², Antonio Molinaro^1*, and José-María Vinardell^2*

¹Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy

²Department of Microbiology, Faculty of Biology, University of Seville, Sevilla, Spain

³Deparment of Organic Chemistry, Faculty of Chemistry, University of Seville, Sevilla, Spain

⁴Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y

Naturales, Universidad Nacional de Río Cuarto-INBIAS, CONICET, Argentina

⁵Istituto per i Polimeri, Compositi e Biomateriali IPCB, Consiglio Nazionale delle Ricerche,

Catania, Italy

*Corresponding authors: José-María Vinardell and Antonio Molinaro

E-mail: jvinar@us.es; molinaro@unina.it

Running title: Sinorhizobium fredii HH103 LPS structure and symbiosis

Keywords: Bacteria, Sinorhizobium fredii, symbiosis, lipopolysaccharide (LPS), pseudaminic

acid, carbohydrate structure, antibody, soybean, Macroptilium, Cajanus

1

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).

Abstract

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

wild-type strain. Also, we previously

demonstrated that the HH103 rkpLMNOPQ

In addition to Nod factors, different

rhizobial surface polysaccharides (RSP) are

commonly necessary for successful

nodulation. Exopolysaccharides (EPS),

operon

is

responsible

for

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

and containing β-configured 5-acetamido-

3,5,7,9-tetradeoxy-7-(3-

hydroxybutyramido)-L-glycero-L-manno-

nonulosonic acid [β-Pse5NAc7(3OHBu)].

This pseudaminic acid derivative, in its α-

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 monoclonal

antibody, 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, this

rkpM mutant was severely impaired in

symbiosis with its host Macroptilium

atropurpureum.

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 rhizobia-legume symbionts, one or more

RSP could act as signal molecules

promoting bacterial infection and/or

preventing plant defence 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. α-

configured 5-acetamido-3,5,7,9-tetradeoxy-

7-(3-hydroxybutyramido)-L-glycero-L-

manno-nonulosonic

acid

(α-

Pse5NAc7(3OHBu)) (12), while 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. A S. fredii Rif^RexoA 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^Rmutants

Introduction

Rhizobia are soil bacteria able to

stablish

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

2

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-

α-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 or rkpQ) did

not produce the α-Pse5NAc7(3OHBu) KPS

as well, showed altered LPS electrophoretic

profiles and only induced the formation of

soybean pseudonodules unable to fix

nitrogen (17).

the infection event, nodule development or

in the bacteroid differentiation, and this

phenomenon has been observed with both

determinate- and indeterminate-nodule

forming legumes (32-36).

In this work, we focused the

attention on the LPS isolated from two S.

fredii HH103 Rif^Rmutants: 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,thus 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 showed to be only

slightly altered with respect to S. fredii

HH103 Rif^R. Structural investigation

showed that HH103 rkpA possesses an LPS

containing β-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 derivative, and that

this mutant is severely impaired in symbiosis

with the HH103 host legume Macroptilium

atropurpureum.

LPSs

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) which is attached

to an outer polysaccharide termed O-chain

(18). On the basis of the nature of the

saccharide portion, an LPS can be classified

as smooth, semi-rough or rough: smooth-

type LPS (S-LPS) contains all the three

above mentioned domains, whereas a semi-

rough-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

Results

Silver-staining and antibody recognition of

the LPS produced by different S. fredii

HH103 mutants affected in LPS and/or

KPS biosynthesis

biovar

viciae

128C53

(19)

and

Bradyrhizobium 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

In previous works (17,31,37), we

have studied different S. fredii HH103

mutants (in the rkpM, lpsB, lpsE, rkpK, and

lpsL genes) which 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 constitutes the so-called rkp-2 region

and their encoded products are responsible

3

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. Wild-type S. fredii HH103

Rif^Rand its rkpA mutant derivative were also

included as controls, since this mutant does

not produce KPS but its LPS shows the same

electrophoretic profile of HH103 Rif^R(16).

Fig. 1A allowed us to compare the

electrophoretic alterations exhibited by the

different mutants. As shown in this figure,

the LPS profile of S. fredii HH103 Rif^Ris

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 disperse 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. Also,

at the bottom of each lane appears a diffuse

band that it is not always visualized in SDS-

PAGE gels carried out with HH103 LPS

samples (16,17) and whose nature is

unknown.

antibody 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^Rwas 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^Rmutants showing

alterations in their LPS electrophoretic

profiles (rkpM, lpsL, rkpK, lpsE, and lpsB)

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 wild-

type LPS profile (Fig. 1A) and recognition

by the monoclonal antibody NB6-228.22

(Fig. 1B).

LPS profiles of S. fredii HH103 Rif^R

free-living cells is undistinguishable from

HH103 Rif^Rbacteroids isolated from G. max

or C. cajan (39). However, S. fredii HH103

Rif^Rbacteroids isolated from Glycyrrhiza

uralensis showed altered LPS profiles in

comparison to 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^Ror

HH103 rkpM were also analyzed by SDS-

PAGE in the present study (Fig. 1A). The

LPS profile of HH103 rkpM 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 in comparison to

those of the wild-type strain. The LPS of

HH103 rkpM bacteroids isolated from

Glycyrrhiza nodules was neither recognized

by the monoclonal antibody NB6-228.22

(Fig. 1B).

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^Ror its

rkpA derivative. However, there were also

differences among the different LPS mutants

(Fig. 1A): mutants in lpsL, rkpK, and lpsE

showed similar LPS profiles with faster

electrophoretic mobility than that of HH103

rkpM but slower than that of HH103 lpsB.

Following these observations, we

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

In order to define the detailed

structure of S. fredii HH103 rkpA and

employed NB6-228.22,

a

monoclonal

4

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 glycosides

derivatization, revealed the presence in both

LPSs of D-galacturonic (D-GalA) and D-

glucuronic (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

LPSs, 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-substituted D-GalpA,

terminal and 2-substituted D-GlcpA, 2-

substituted and 3-substituted D-Glcp, 3,6-

to chemical analyses. The analysis of both

the ¹H NMR (Fig. 2) and HSQC spectra (Fig.

3) of the pure OS1_rkpAclearly showed the

occurrence of eight anomeric signals

attributed to eight different spin systems (A-

H, Table 2), while the presence of a Kdo unit

was attested by the presence of the

diasterotopic H-3 methylene proton signals

at _H1.94/1.75 ppm (K, _C33.9 ppm, Figs. 2

and 3). Furthermore, the ¹H NMR spectrum

(Fig. 2) showed the presence of four intense

methyl signals at _H1.05, 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^IH-3_ax/H-3_eq, _C36.0

ppm,) and 1.50/2.47 ppm (Pse^IIH-3_ax/H-3_eq,

_C36.1 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 (hereafter indicated as

Pse^Iand Pse^II). Briefly, the diasterotopic

methylene protons H-3 Pse^Ishowed long-

range correlations with an oxymethine

carbon atom (C-4 Pse^I, _C66.3 ppm), a

quaternary carbon (C-2 Pse^I, _C103.2 ppm)

and a carboxylic group (C-1 Pse^I, _C172.8

ppm) (Fig. 4). A less intense HMBC

correlation between H-3_axPse^Iand 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^Iwas 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 nitrogen-bearing 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-hydroxybutyryl moiety (HBu,

made up of the CH₃-C group at _C22.0 ppm,

the C-CHOH-C group at _C69.8 ppm, the C-

CH₂-C at _C45.0 ppm, and the CO group at

173.4 ppm) was found to be connected by

amide linkage at position 7 of Pse^I; the

disubstituted

D-Galp,

6-substituted-D-

GlcpN, terminal and 4,5-disubstituted Kdo

were detected in both the LPSs. Importantly,

as described further in the text, an additional

sugar constituent, the Pse5NAc7(3OHBu)

(Pse) was identified solely in HH103 rkpA

and only after an in-depth investigation of

the isolated LPS saccharide component

using NMR spectroscopy and MALDI-TOF

mass spectrometry (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 in

order 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

high-pressure liquid chromatography, the

pure saccharide moieties (OS1_rkpAand

OS1_rkpM) were analyzed via 1D and 2D

NMR spectroscopy including DQF-COSY,

1

TOCSY, ROESY, H-¹³C HSQC, H-¹³C

HSQC-TOCSY and ¹H-¹³C HMBC.

All sugar units were present as

pyranose rings, according to the ¹³C

chemical shift values and in full accordance

5

location of the HBu moiety was attained by

the observation of a long-range correlation

between C-1' (173.4 ppm) and H-7 Pse^I

(3.94 ppm) in the HMBC spectrum (Fig. 4).

Due to the relatively large J_H-3ax,H-4(∼12.9

Hz) and the small J_H-4,H-5and J_H-5,H-6(<5 Hz)

coupling constants it was possible to deduce

the axial orientation of H-4 Pse^Iand the

equatorial orientation of H-5 Pse^I, that

combined with the J_H-6,H-7coupling 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, by

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_axand H-3_eqchemical shifts

(0.88 ppm) indicated the axial orientation of

³J_H-1,H-2values and the NOE contacts of H-1

with H-3 and H-5. Therefore, A and B were

identified as α-GalA units, C as an α-GlcA,

F as a β-GalA, and H as a β-GlcA. Residue

D (H-1 at 5.04 ppm, Table 2) was identified

3

as α-Gal owing to the small J_H,Hvalues 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 α- and β-Glc respectively.

Low-field shifted carbon signals

(Table 2 and Fig. 3) were useful to identify

substitution at O-4 of residues A and B, at O-

2 of F and H, at O-3 and O-6 of D, at O-3

of G, at O-4 and O-5 of K, and, as described

above, at O-3' of Pse^I; on the other hand,

residues C, E and Pse^IIwere 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 inter-residue 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^Iand H-3' Pse^I/C-2 Pse^II(Fig. 4)

were identified and led to the structural

assessment of the OS1_rkpAproduct (Fig. 5).

In order 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 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 due to

the harsh alkaline treatment and the intrinsic

nature of the oligosaccharide, the NMR

the carboxyl group, that is the

β

configuration of Pse^Iat the anomeric center

(42). Finally, the downfield displacement of

the chemical shift of C-3' (69.8 ppm) of unit

Pse^Isuggested a site of glycosylation at such

a position. Similarly, a second unit of β-

Pse5NAc7(3OHBu) has been assigned

(Pse^II) but it turned out to be a terminal

residue, linked to position 3' of Pse^I, as

proven by the long-range correlation

between the C-2 of Pse^IIand the oxymethine

proton H-3' (4.13 ppm, Table 2 and Fig. 4)

of Pse^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 due to 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.

analysis of the obtained product (OS2_rkpA

)

3

clearly disclosed, in addition to the signals

relative to the α-GlcN (X) and β-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

(_C33.9 ppm). Finally, the observation of the

low-field position of the signal for C-2 (75.4

ppm) of the α-Glc (E₂) linked to the 4,5-α-

Kdo (K₂), and the occurrence of a NOE

correlation between H-3 J and H-2 of E₂led

to the determination of the sequence J-

(2→2)-E₂-(1→4)-K₂, with the latter Kdo

The J_H-3,H-4and J_H-4,H-5coupling constant

values (3 and 1 Hz, respectively) measured

for A, B and F were diagnostic of a galacto

3

configuration whereas the large ring J_H,H

coupling constants observed for C and H led

to their identification as gluco-configured

sugar units. The α-anomeric configuration

for A, B and C was attributed on the basis of

the intra-residual NOE contact of H-1 with

H-2, and the ³J_H1,H2coupling constant values,

whereas the β-anomeric configuration for

residues F and H was inferred by the large

6

unit as the one linked to β-GlcN (Y) of the

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 β-Pse5NAc7(3OHBu), two

Kdo units, one α-D-Glcp, one D-β-Glcp, one

D-α-Galp, two D-α-GalpA, one D-β-GalpA,

one D-α-GlcpA and one D-β-GlcpA, as

sketched in Fig. 5.

ranges (m/z 1500-2100). This LPS

fragmentation (β-elimination) yielding both

oligosaccharide(s) (OS) ions and lipid A

ions furnished important details about the

structure of HH103 rkpA LPS. In particular,

the MALDI-TOF MS investigation gave the

possibility to appreciate the heterogeneity of

both the lipid A and the core OS portion of

such LPS. Indeed, the main ion fragments in

Fig. 6A at m/z 1770.2 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), thus 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.

A similar approach was used to

elucidate the structure of OS1_rkpM(Table

S1). The comparison of the NMR spectra of

OS1_rkpMand 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 ¹H- and the HSQC NMR

spectra (Fig. 2 and S2) no signals relative to

the methylene and methyl groups of Pse

residues were visible. Consequently, the β-

GalA unit F' of OS1_rkpM(Table S1),

corresponding to residue F in the rkpA

mutant, was identified as a terminal sugar

due to 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.

Also 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).

Similarly, the reflectron MALDI-

TOF MS spectrum of intact LPS from

HH103 rkpM mutant revealed the

occurrence of a main fragment ion at m/z

1409.5 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/z 1629.7, OS_B) and HexA (m/z 1805.5,

OS_C) have also been identified. Therefore,

by MALDI-TOF MS analysis it was possible

to further confirm that HH103 rkpM

expresses an LPS devoid of Pse, according

to NMR data. As described for rkpA mutant,

in the higher mass region LPS species

composed by the above OS species and

differently acylated lipid A forms have been

detected.

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

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),

7

28:0(27-OHBu),

and 30:0(29-OHBu),

rkpA but absent in rkpM. Since NB6-228.22

binds to the LPS produced by rkpA but fails

in the recognition of rkpM LPS, we

investigated whether this monoclonal

antibody was also able to recognize the KPS

produced by HH103 Rif^R, which is a

homopolymer of α-Pse5NAc7(3OHBu)

(12).

which is consistent with the lipid A fatty acid

composition from the genus Sinorhizobium

(23). In order to unveil the structure of the

glycolipid portion of the LPS from both

mutants, an aliquot of the mild acid

hydrolysis precipitated product, containing

the lipid A, has been analyzed by negative-

ion MALDI-TOF MS (the spectrum is

reported in Fig. S3A, B). The MALDI mass

spectra of the isolated lipid A fractions were

identical for both mutants and further

confirmed, and expanded, 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 example, in the

higher masses region the ion peak at m/z

For this purpose, intact cells of S.

fredii HH103 Rif^Rand 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 immuno-stained 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 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.

2066.0 was attributed to

phosphorylated penta-acylated lipid

a

bis-

A

species carrying two 14:0(3-OH), one

18:0(3-OH), 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/z 1952.0 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 peak at m/z

1725.9. At m/z 2050.0 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/z 1743.9. The more

intense ion peak at m/z 1870.0 was assigned

to a 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/z 1643.9.

The epitope recognised by the monoclonal

antibody 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).

The monoclonal antibody NB6-228.22

recognizes S. fredii HH103 Rif^RLPS but

fails in the recognition of KPS

Structural

demonstrated that

investigation

β-configured

a

Pse5NAc7(3OHBu) is present in the LPS of

8

Immuno-staining 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).

and C. cajan, which form determinate and

indeterminate nodules, respectively (17,38).

In the case of M. atropurpureum

(Table 3 and Fig. 9), HH103 rkpA, affected

in KPS but not in LPS production, was also

included in these studies. The number and

fresh weight of nodules of M.

atropurpureum plants inoculated with

HH103 rkpA or HH103 rkpM were similar

and significantly lower than those formed on

plants inoculated with HH103 Rif^R, although

nodules induced by HH103 rkpM were

white. Plants inoculated with these mutants

were stunted with yellow leaves and their

plant-top dry-weight was not significantly

different from that of the uninoculated

control. In contrast, plants inoculated with

HH103 Rif^Ror HH103 rkpM C produced

green leaves and plants did not show

Since 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, and

rkpQ genes using genomic DNA (gDNA) as

template (Tables S3 and S4). Strain NGR234

was not included in these experiments since

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

symptoms

of

nitrogen

starvation.

Nitrogenase activity (nmol ethylene/3 plants

x hour, determined by ARA, acetylene

reduction assays) was similar in plants

growing in the three jars inoculated with

HH103 Rif^R(136 ± 43) or HH103 rkpM C

(154 ± 63), and lower in the three jars

inoculated with HH103 rkpA (36 ± 14).

When HH103 rkpM was used as inoculant, a

very low nitrogenase activity could be

detected and only in two out of the three jars

analyzed (4.7 and 15.1). Microscopy

analyses showed dispersed patches of nodule

cells invaded by HH103 rkpM (Fig. 10A2),

which contained less bacteroids than those

infected by S. fredii HH103 Rif^Ror HH103

rkpM C (Fig. 10A1 and 10A3). Nodules

induced by HH103 rkpA (Fig. 10A4) showed

an infection level that could be considered as

an intermediate stage between those induced

by HH103 Rif^Rand HH103 rkpM.

an identity of 98-100

%

with the

corresponding rkp gene of S. fredii HH103

Rif^R(Table S4).

Since S. fredii S50 appeared to have

only part of the rkpLMNOPQ operon, we

investigated the presence of these genes in

sinorhizobial strains for which homologous

sequences (>85.0 identity at aminoacid

level) to RkpM were found in silico. The

complete operon was present in S. fredii

CCBAU05631, Sinorhizobium sp. 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 was missing. S.

fredii strain CCBAU45436 lacks rkpO and

the rest of predicted proteins showed

identities lower than 55 %. Fig. 8 shows the

organization of the rkp-3 region in S. fredii

strains as well as in S. meliloti Rm41.

The symbiotic impairment of

another rkpM mutant (called SVQ582) with

C. cajan was previously reported (17),

although microscopy of the pseudonodules

produced was not carried out. Since infected

cells were found in M. atropurpureum

nodules, we investigated whether C. cajan

pseudonodules contained cells invaded by

the HH103 rkpM mutant investigated in the

present work. Plant-top dry-weight of C.

cajan plants inoculated with HH103 rkpM

was not significantly different from that

shown by uninoculated controls (78 and 101

mg/plant, respectively). Plants inoculated

The symbiotic impairment of the S. fredii

HH103 rkpM mutant is higher with

Cajanus cajan than with Macroptilium

atropurpureum

The symbiotic relevance of LPS and

KPS in rhizobia-legume interactions has

been extensively documented (1-5). In this

work, we have analyzed the symbiotic

phenotype of HH103 rkpM and its

complemented version HH103 rkpM C with

two HH103 host legumes, M. atropupureum

with

HH103

rkpM

only

formed

9

pseudonodules and macroscopic swellings

(Table 3, Fig. 9). Microscopy images

showed that pseudonodules induced by this

mutant were devoid of bacteroids (Fig.

10B2). C. cajan plants inoculated with S.

fredii HH103 Rif^Ror HH103 rkpM C formed

nitrogen-fixing nodules, plants were green

and did not show symptoms of nitrogen

starvation (Fig. 9).

strains (23). The lack of function of RkpM

also affects to the alterations that the S. fredii

HH103 LPS undergoes during bacteroid

development in nodules of Glycyrrhiza

uralensis.

The HH103 lpsB, lpsE, lpsL and

rkpK mutants are also presumably affected

in the core structure of their LPS. LpsB and

LpsE are predicted glycosyl transferases

(31), whereas LpsL and RkpK are

respectively responsible for the synthesis of

GalA and GlcA (37), and, as demonstrated

in the present work, the HH103 LPS core

contain hexoses and these two uronic acids.

We planned to analyse the LPS structure of

these four HH103 LPS mutants in the next

future.

Discussion

In this work we report the

characterization of the full structure of the

LPS from S. fredii HH103 rkpA. We select

this mutant because its LPS shows the same

electrophoretic profile and reactivity with

the monoclonal antibody NB6-228.22 as the

wild-type strain LPS does. Thus, the LPS

structure of HH103 rkpA is assumed to be

equal to that of the wild-type strain. In

addition, HH103 rkpA is devoid of KPS, a

polysaccharide that can interfere with LPS

purification procedures. Our work reveals a

novel core OS with a high content in

hexuronic acids and showing the presence of

β-Pse5NAc7(3OHBu) located at the

outermost part of the LPS. This residue was

found to be linked to another one of the same

nature, which might constitute an extremely

short O-chain moiety (Fig. 5). In previous

works, we obtained different HH103

mutants affected in LPS structure as

reflected in their altered electrophoretic

profiles (Table 1) (17,31,37). In this work

we have also carried out the structural

analysis of the LPS produced by HH103

rkpM, since its LPS electrophoretic profile

was the less altered, compared to that of

HH103 LPS, among the different HH103

mutants available. Our work highlights that

the core OS of HH103 rkpM lacks

Pse5NAc7(3OHBu) (Fig. 5). This result is in

full accordance with the fact that the

rkpLMNOPQ operon is involved in the

biosynthesis of Pse derivatives in S. meliloti

41 (47) and S. fredii HH103 (17). Finally, the

lipid A moieties of both rkpA and rkpM were

found identical and displayed a high degree

of heterogeneity in the acyl chains which

The monoclonal antibody NB6-

228.22 recognizes all the LPS silver-stained

bands of wild-type HH103 and its rkpA

mutant derivative (unable to produce KPS)

as well as entire cells of these two strains and

a purified HH103 LPS sample. However, it

failed to perceive the LPS of HH103 rkpM,

which strongly suggested that the

Pse5NAc7(3OHBu) residue present in the

LPS core OS of HH103 and HH103 rkpA

should be the epitope (or part of it)

recognized by this antibody. Interestingly,

antibody NB6-228.22 failed to perceive a

purified sample of the S. fredii HH103 KPS,

regardless Pse5NAc7(3OHBu) is the only

component of this polysaccharide, as well as

failed in the recognition of intact cells and

LPS profiles of HH103 lpsL, lpsB, lpsE, and

rkpK mutants (altered LPS, wild-type KPS).

Thus, it was possible to conclude that the

monoclonal

antibody

NB6-228.22

recognizes the HH103 LPS but not its KPS.

There are various possibilities that could

explain why the same monosaccharide,

Pse5NAc7(3OHBu), is recognized in the

LPS but not in the KPS. One very likely

explanation is that Pse5NAc7(3OHBu)

residues are α-linked in the KPS (12) but β-

linked in the LPS. Alternatively, the epitope

perceived by the antibody might not be

restricted to only Pse5NAc7(3OHBu),

which is the case of the HH103 Rif^RKPS but

not of the LPS in which Pse5NAc7(3OHBu)

is linked to a D-GalA residue (Fig. 5). In any

case, the epitope recognized by the antibody

NB6-228.22 is poorly frequent in S. fredii

strains as demonstrated by the fact that only

2 out of 25 strains analyzed (including

were

stoichiometric

characterized

by

the

non-

by

substitution

hydroxybutyrate on the VLCFA and by the

occurrence of unsaturated chains, as

previously reported for other Sinorhizobium

10

HH103) reacted with this antibody. This fact

is not unexpected since the LPS core and O-

antigen regions are known to vary at the

strain level (1,19).

nitrogen starvation (14-17). Considering all

these results we can conclude that in S. fredii

HH103 the combination of absence of KPS

production and an alteration of the LPS,

PCR experiments and in silico

analyses carried in this work showed that the

consisting

in

the

absence

of

Pse5NAc7(3OHBu), causes the most severe

symbiotic impairment with these two

legumes. A S. fredii HH103 Rif^Rdouble

(rkpH and exoA) mutant unable to produce

KPS and EPS (LPS electrophoretic profile

apparently unaffected) was still able to

induce the formation of some nitrogen fixing

nodules with G. max cvs. Williams and

Peking (14). Thus, the loss of EPS

production in a S. fredii HH103 unable to

produce KPS is not so harmful for the

bacterial symbiotic capacity with soybeans

than the simultaneous absence of KPS

production and Pse5NAc7(3OHBu) residues

in the LPS structure.

rkpLMNOPQ

operon

involved

in

Pse5NAc7(3OHBu) biosynthesis can be

found in diverse Sinorhizobium species but

its presence might not be frequent at strain

level. In addition, some of the Sinorhizobium

strains containing the region lack some of

these genes. S. fredii S50 amplified the rkpM

and rkpP genes but failed in the

amplification of rkpQ and rkpL. The

amplified internal sequences of S50 rkpM

and rkpP are nearly identical to those of

HH103 (98-100 % identity), which suggests

that the failure to amplify rkpQ and rkpL

might be due to the absence of these genes in

S50 rather than to unsuitable nucleotide

sequences of the primers used. S. fredii

NGR234 and S. saheli LMG7837 are devoid

of orthologues of rkpP, and S. fredii

CCBAU45436 lacks rkpO. The absence of

rkpP does not prevent the biosynthesis of the

Pse derivative present in the NGR234 KPS

(46).

Thus, two types of plant responses to

inoculation with HH103 rkpM can be

observed in both determinate- and

indeterminate-nodules forming legumes: G.

max, V. unguiculata, and C. cajan formed

uninfected pseudonodules while L. burttii,

M. atropurpureum, and G. uralensis were

still able to form a variable number of

nitrogen fixing nodules. All these results

indicate that the symbiotic impairment of

mutations affecting S. fredii HH103 surface

polysaccharides vary among the different

legumes tested, regardless they form

indeterminate or determinate-nodules and

that the simultaneous alteration of two

polysaccharides can produce unpredictable

impacts on the bacterial symbiotic capacity.

RSP, such as LPS and KPS, can be

In previous studies we showed that

two plants forming determinate nodules,

soybean and cowpea (Vigna unguiculata),

when inoculated with HH103 rkpM only

formed pseudonodules that did not fix

nitrogen and were devoid of intracellular

bacteria, whereas this mutant was able to

induce the formation of Fix+ nodules with

Lotus burttii (determinate nodules) and

Glycyrrhiza

uralensis

(indeterminate

nodules) (17,48). In this work, we have

also important for stress tolerance both in

free-living conditions and during the

infection process and bacteroid persistence

(29,31,49). Here we have studied the effect

of the rkpA and rkpM mutations on the final

growth (expressed as OD₆₀₀scored 24, 48 or

120 hours after inoculation) of S. fredii

HH103 in liquid minimal medium

supplemented with high concentrations of

either sodium chloride (100 o 150 mM) or

the detergent SDS (0.35 or 0.52 mM) (Fig.

S4). No differences among the different

strains were found in the presence of 100 or

150 mM NaCl or SDS 0.35 mM. However,

five days after inoculation, the growth of S.

fredii HH103 Rif^Rand HH103 rkpM C in

liquid MM supplemented with 0.52 mM SDS

included

forming

another

legume,

determinate-nodule

Macroptilium

atropurpureum, and showed that nodulation

of plants inoculated with HH103 rkpM was

severely reduced and nitrogen fixation

almost abolished in comparison to those

inoculated with HH103 (Table 3).

In contrast to HH103 rkpM, S. fredii

HH103 lpsB and lpsE mutants (presenting

KPS but with an altered LPS) as well as S.

fredii HH103 mutants in genes of the rkp-1

region (unable to produce KPS but with a

normal LPS) are still able to induce the

formation of Fix⁺nodules in both soybean

and cowpea plants, albeit nodulation is

reduced and plants show symptoms of

11

was higher (OD₆₀₀>0.8) than that observed

for HH103 rkpM (OD₆₀₀about 0.3) or

HH103 rkpA (OD₆₀₀about 0.5) (Fig. S4).

These results suggest that both KPS and LPS

apparently provide protection against SDS.

Whether these phenotypes are related to the

symbiotic impairment exhibited by HH103

rkpM requires further investigation.

staining, and immuno-staining procedures

with the monoclonal antibody NB6-228.22

(Class IgG 2b) were performed as described

previously (38). Production of the

monoclonal antibody NB6-228.22 (Class

IgG 2b) is described by Buendía-Clavería

and collaborators (38). Briefly, soybean

nodule-derived bacteroids of HH103 were

used to immunize male rats (Line LOU/IAP)

and spleen cells derived from these rats were

fused to the myeloma line IR983F. NB6-

228.22, which recognises all the LPS bands

from HH103 free-living cells or HH103

bacteroids isolated from soybean nodules.

NB6-228.22 was purified from the

hybridoma culture supernatant.

In conclusion, with this work a novel

LPS structure has been identified revealing

the occurrence of an unprecedented

component for rhizobial LPS, a β-configured

Pse5NAc7(3OHBu), whose presence might

be important for the establishment of an

effective symbiosis of S. fredii HH103 with

some of its host plants. This crucial point

will be object of future structure to function

studies focused on the determination of the

molecular motifs of S. fredii HH103 LPS

that may be crucial for bacteria–plant cell

interactions.

Binding assays of intact S. fredii

cells to NB6-228.22 were carried out as

follows: a loop of S. fredii cells grown on

solid TY for four days was suspended in 200

µL of sterile water. Then, 10 µL drops of

bacterial suspensions were deposited on

positively

charged

Nylon

transfer

membranes (Amersham Bioscience, UK).

Intact bacterial samples absorbed to the

filters were immunostained with NB6-

288.22, using as the secondary antibody a

goat anti-rat IgG conjugated to alkaline

phosphatase (Sigma).

Experimental procedures

Extraction, purification, and SDS-PAGE

analysis of rkpA and rkpM LPS

Dried bacterial cells of HH103 rkpA

and rkpM were washed several times with

distilled water, ethanol, acetone and ethylic

ether

to

remove

growth

culture

Chemical analyses of HH103 rkpA and

rkpM LPS

contaminants. After lyophilization, the

bacterial pellets were extracted by the

phenol/water method (40). After extensive

dialyses against distilled water, the extracted

phases were suspended in 40 mL of

digestion buffer (100 mM Tris, 50 mM NaCl,

10 mM MgCl₂, pH 7.5) to which RNAse (4

mg, R5503-Merck), DNAse (4 mg, DN25-

Merck) and proteases (4 mg) were added to

remove nucleic acids and protein

contaminants. Both the water and phenol

fractions were checked by SDS-PAGE after

silver staining; the LPS material, in both

HH103 rkpA and rkpM, was found

exclusively in the water phases. To further

purify the samples, the LPSs were

ultracentrifuged (4 °C, 208,000 × g, 16 h)

and then subjected to size-exclusion

chromatography on a Sephacryl S-300 (GE

Healthcare, 1.5 x 90 cm, eluent NH₄HCO₃

50 mM).

Monosaccharides were identified as

acetylated O-methyl glycoside derivatives

obtained by methanolysis (1.25

M

HCl/MeOH, 85 °C, 16 h) followed by

acetylation with acetic anhydride in pyridine

(80 °C, 30 min) (41). To define the sugar

linkage pattern, including uronic acids and

Kdo, an aliquot of each sample was

methylated with iodomethane, treated with

sodium tetradeuteroborate (NaBD₄) to

transforms the methyl ester functions of

acidic sugars in a hydroxymethyl group with

two deuterium atoms, then hydrolyzed with

trifluoroacetic acid (2 M, 100 °C, 2 h),

carbonyl-reduced by employment again of

NaBD_4,acetylated with acetic anhydride and

pyridine, and analyzed by GLC–MS (41,

50). The absolute configuration of sugar

residues has been determined by GLC–MS

analysis of the acetylated O-(+)-2-octyl

glycosides derivatives and comparison with

authentic standards (41, 51).

LPS extraction for PAGE and

immune-staining analyses, as well as the

separation on SDS-PAGE gels, silver-

12

The total fatty acid content was

established by treating each LPS with 4 M

HCl (100 °C, 4 h) and followed by a

treatment with 5 M NaOH (100 °C, 30 min).

The pH was adjusted to reach slight acidity.

Fatty acids, after extraction in CHCl₃, were

methylated with diazomethane and analyzed

by GLC-MS. The ester bound fatty acids

were selectively released by base-catalyzed

hydrolysis with aqueous NaOH 0.5 M,

MeOH (1:1, v/v, 85 °C, 2 h), and then the

product was acidified, extracted in

chloroform, methylated with diazomethane,

and analyzed by GLC-MS. Moreover, an

aliquot of each LPS fraction (0.5 mg) was

also methanolized with 1.25 M HCl/CH₃OH

(80 °C for 16 h). The mixture underwent

three extractions with hexane. The hexane

layer, containing the fatty acids as methyl

esters derivatives, was then analyzed by

GLC-MS.

column (Supelco-Sigma, run in NH₄HCO₃

50 mM at 0.8 mL min^-1, mounted on Agilent

1100 system equipped with a refractive

index and an UV detector, this last set at 206

nm).

Isolation of the oligosaccharides OS2_rkpA

and OS2_rkpM

In order to remove O-linked acyl

chains, an aliquot of each sample was treated

with anhydrous hydrazine, stirred at 37 °C

for 60 min, cooled, poured into ice-cold

acetone, and allowed to precipitate. The

precipitate was centrifuged (1,200 × g, 30

min), washed with cold acetone in an ice-

bath, dried, dissolved in water and

lyophilized (52). The O-deacylated product

was then N-deacylated with KOH (4 M, 120

°C, 16 h). After adjustment of the pH, free

fatty acids were removed by several

extractions with CHCl₃followed by

centrifugation (1,200 × g, 30 min). The

aqueous phase of each sample was then

lyophilized and the produced salts were

removed by size-exclusion chromatography

on a Sephadex G-10 column (Pharmacia, 50

x 1.5 cm, eluent distilled water) to yield

OS2_rkpAand OS2_rkpMfractions.

All the above derivatives were

analyzed on Agilent Technologies Gas

Chromatograph 6850A interfaced with Mass

Spectrometry: a selective Mass Detector

5973N equipped with a SPB-5 capillary

column (Supelco, 30 m x 0.25 mm i.d., flow

rate 0.8 mL min^-1using He as gas carrier).

Electron impact mass spectra were recorded

with an ionization energy of 70 eV and an

ionizing current of 0.2 mA

NMR spectroscopy of the isolated OS1 and

OS2

1

1D and 2D H NMR spectra were

recorded on a Bruker 600 DRX equipped

with a cryo probe. The solvent employed

was D₂O and the temperature was 298 K and

pD was 7. To obtain information on the NH

chemical shifts the experiments were

recorded in an H₂O-D₂O mixture (53).

Spectra calibration was performed with

internal acetone (δ_H2.225 ppm, δ_C31.45

ppm). The double-quantum filtered phase

sensitive correlation spectroscopy (DQF-

COSY) experiment was carried out by using

data sets of 4096 x 256 points. Total

Isolation of the lipid

oligosaccharides OS1_rkpAand OS1_rkpM

A

and the

The OS1 fractions of the two

HH103 mutants investigated were obtained

by mild acid hydrolysis. Purified LPS (~10

mg) was dissolved in acetate buffer (1 mL,

pH 4.4). SDS (1 mg mL^-1) was added, and

hydrolysis proceeded (100 °C, 2 h). The

solutions were extracted three times with

CHCl₃/MeOH/H₂O (100:100:30, v/v/v) and

centrifuged (4 °C, 8,800 × g, 20 min). The

organic phases containing the lipid A moiety

were further purified by several washes with

distilled water, lyophilized and then

analyzed by MALDI-TOF MS. The water

phases, containing the OS1 fractions, were

recovered and lyophilized. These OS1

fractions were then purified by gel filtration

chromatography on a Bio-Gel P-4 column

(Bio-Rad, 1.5 x 100 cm, eluent distilled

water) and then, once dissolved in

NH₄HCO₃50 mM, further purified via

HPLC by using a TSKgel G5000PWXL

correlation

spectroscopy

(TOCSY)

experiments were executed with spinlock

times of 100 ms, using data sets (t1 × t2) of

4096

x

256 points. Rotating frame

Overhauser enhancement spectroscopy

(ROESY)

enhancement

and Nuclear

spectroscopy

Overhauser

(NOESY)

experiments were recorded by using data

sets (t1 × t2) of 4096 x 256 points and by

using mixing times between 100 and 400 ms.

In all homonuclear experiments the data

13

matrix was zero-filled in both dimensions to

give a matrix of 4 K x 2 K points and was

resolution enhanced in both dimensions by a

cosine-bell function before Fourier

Transformation. The determination of

coupling constants was obtained by 2D

phase sensitive DQF-COSY (54,55).

Heteronuclear single quantum coherence

(¹H- ¹³C HSQC) and heteronuclear multiple

bound correlation (¹H- ¹³C HMBC)

spectrometer. Lipid

dissolved in CHCl₃/CH₃OH (50:50, v/v).

The matrix solution was 2,4,6-

trihydroxyacetophenone in CH₃OH/0.1 %

trifluoroacetic acid/CH₃CN (7:2:1, v/v) at a

concentration of 75 mg mL^-1(60). 0.5 μL of

the sample and 0.5 μL of the matrix solution

were deposited on a stainless-steel plate and

left to dry at room temperature.

A fractions were

1

experiments were recorded in H-detection

Molecular and microbiological techniques

Bacterial strains and plasmids used

mode by single-quantum coherence with

proton decoupling in the ¹³C domain using

in this work are listed in Table S6. S. fredii

strains were grown at 28 ºC on complete TY

medium (61), liquid YMB (62), or liquid

MM (63). YMA medium corresponds to

YMB solidified with agar (20 g/L). When

necessary, the media were supplemented

with antibiotics (concentrations in µ mL^-1):

rifampicin (Rif) 25; tetracycline (Tc), 2-4

(10 for Escherichia coli); gentamycin (Gm),

5 (10 for E. coli); spectinomycin (Spc), 50 or

100.

1

data sets of 2048 × 256 points. H- ¹³C

HSQC was executed using sensitivity

improvement and in the phase-sensitive

mode using Echo/Antiecho gradient

selection, with multiplicity editing during

1

selection step (56). The H- ¹³C HMBC

experiment was optimized on long-range

coupling constants with low-pass J filter to

suppress one-bound connectivity, using

gradient pulses for selection. A delay of 60

ms was employed for the evolution of long-

range correlations. It was used a long-range

coupling constant value of 6 Hz. The data

matrix in both heteronuclear experiments

was extended to 2048 × 1024 points using

forward linear prediction (57).

Assays to determine bacterial

tolerance to SDS were carried out in MM

medium containing 0.35 or 0.52 mM SDS. A

50 mM SDS stock solution in water was

sterilized by filtration (0.2 µm pore size) and

added to sterile MM to reach final

concentrations of 0.35 or 0.52 mM SDS.

Aliquots (50 µL) of ten-fold dilutions of

MALDI-TOF MS of intact LPS from

HH103 rkpA and rkpM and their isolated

lipid A fractions

early-stationary

bacterial

cultures

(OD₆₀₀=1.0) were used to inoculate tubes

containing 5 mL of MM supplemented with

the detergent. Bacterial growth was

spectrophotometrically monitored at 1, 2,

and 5 days after inoculation. Similarly,

assays to determine salt tolerance were

carried out in MM media supplemented with

100 or 150 mM NaCl.

MALDI-TOF mass spectra of intact

LPS from HH103 rkpA were recorded in

linear mode and negative ion polarity on an

Applied Biosystems MDS SCIEX 4800

MALDI TOF/TOF™ Analyzer, equipped

with delayed extraction technology. Ions

formed by a pulsed UV laser (Nd:YAG

laser, λ= 355 nm) were accelerated by 24 kV.

The MS structural investigation of HH103

rkpM LPS was performed in reflectron mode

and negative ion polarity on an ABSCIEX

TOF/TOF^TM5800 Applied Biosystems mass

spectrometer equipped with an Nd:YAG

laser (λ = 349 nm), with a 3 ns pulse width

and a repetition rate of up to 1000 Hz. In

both cases, LPS MALDI preparations were

executed as previously reported (50, 58, 59).

Reflectron MALDI-TOF MS spectra of lipid

A isolated from both HH103 mutant strains

analyzed in this work were recorded on the

above described AB SCIEX TOF/TOF^TM

PCR amplifications were carried out as

previously described (64). Primers for PCR

amplifications are listed in Table S4.

Nodulation tests

Plant tests were carried out on

Glycyrrhiza uralensis (L), Macroptilium

atropurpureum (Moc. & Sessé ex DC) Urb.,

and Cajanus cajan (L.) Millsp. Inoculated G.

uralensis nodules were used as source of

bacteroids for the visualization of LPS

electrophoretic profiles. M. atropurpureum

and C. cajan plants were used for standard

nodulation assays, in which parameters of

5800

Applied

Biosystems

mass

14

symbiotic significance were determined.

Nodule structures by optic and electronic

microscopy of nodules induced by the

HH103 rkpM mutant were also investigated.

M. atropurpureum and G. uralensis

seeds were surface sterilized by sequential

exposure to sulphuric acid for 20 min and

sodium hypochlorite 12 % (w/s) for 5 min,

followed by five washing steps with sterile

distilled water. C. cajan seeds were

sterilized by sequential exposure to 96 %

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. Then, small

fragments of M. atropurpureum nodules

were immediately fixed in 4 % (v/v)

glutaraldehyde in 0.1 M cacodylate buffer,

pH 7.2 for 2 h at 4 °C after collection.

Samples were washed several times in 0.1 M

cacodylate buffer, pH 7.2, dehydrated in

(v/v) ethanol for 30

s and sodium

hypochlorite 12 % (w/s) for 5 min.

Two germinated seeds of G.

acetone

at

progressively

higher

uralensis or C. cajan were transferred to

individual Leonard jars containing 800 mL

of sterilized vermiculite supplemented with

Fåhraeus nutrient solution (64). Three M.

atropurpureum germinated seeds were

placed in 250 mL Leonard jars. Each

seedling was inoculated with 1 mL of a

particular bacterial culture grown in YMB

(O.D.₆₀₀=0.4-0.5).^.Inoculated plants were

grown for seven (M. atropurpureum) or

eight (C. cajan) weeks with a 16 h

photoperiod at 25 ºC in the light and 18 ºC in

the dark. Nitrogenase activity of M.

atropurpureum nodules was detected by

acetylene reduction assays (ARA) as

described previously (65). Briefly, M.

atropurpureum roots were cut off and

respectively placed in a 10 mL sealed test-

tube or a 250 mL sealed flask. The reaction

was started with an air gas phase containing

10 % acetylene. After incubating for 1 h,

concentrations and embedded in Spurr resine

(low viscosity embedding kit, by Dr. Spurr).

The semi-thin sections (1 µm) were obtained

on a Leica EM UC7 ultramicrotome, stained

with Toluidine blue and viewed in an

Olympus BX61 light microscope.

Extraction of bacteroids 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 mM 3-(4-morpholino)-

propane sulfonic acid, 20 mM KOH, 2 mM

MgSO4, 0.3 M sucrose, pH 7.0) buffer, and

the liquid containing bacteroids was

removed with a pipette. Large particles were

removed by passing through a 40 μm cell

strainer cap (BD Falcon, BD Biosciences,

San Jose, CA, USA). Samples were

centrifuged once at 70 × g for 5 min and

posteriorly twice at 2200 × g for 5 min in

order to re-suspend the resulting pellet

fraction in MMS medium. LPS extraction

from isolated bacteroids was carried out by

following the same protocol used for the

extraction of LPS from free-living cultures

(38).

ethylene was measured with

a

gas

chromatograph (5890A Hewlett Packard)

using a Porapack Q column and nitrogen

carrier gas at 80 ºC. Plant tops were dried at

80 ºC for 48 h and weighted.

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^RGm^Rfor HH103 rkpM and

rkpA mutants, and Rif^RGm^RTc^Rfor HH103

rkpM C.

Data Availability.

The authors state that all the data described

in the manuscript are contained within the

manuscript.

Acknowledgements:

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).

We thank the Centro de Investigación, Tecnología e Innovación (CITIUS) of the University of

15

Seville for light microscopy facilities. MSD is Career Member of CONICET and worked in this

work thanks to grant CONICET PIP 112-201501-00232.

Author contributions

FDL, MARC, JERS, JMV, and AM conceived and designed the experiments. FDL, CAS, SAJ,

MARC, MSD, AP, and DG performed the experiments. FDL, MARC, JERS, JMV, and AM

analysed the data. JERS, JMV, and AM contributed with reagents/materials/analysis tools. FDL,

IS, AS, MARC, JERS, JMV, and AM wrote the paper. All authors have read and approved the

final manuscript.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents

of this article.

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18

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21

Table 1. Previously studied Sinorhizobium fredii HH103 mutants affected in LPS.

Mutated

gene

Predicted function of the

encoded product

Glycosyl transferase

UDP-glucuronate 4-epimerase

UDP-glucose

6-dehydrogenase

Pyridoxal phosphate (PLP)-

dependent aminotransferase

RSP

affected^a

LPS*

Symbiotic phenotype with

soybean

High reduction in the number

of Fix⁺nodules, pseudonodules

Pseudonodules, Fix^–

Reference

lpsB

lpsE

lpsL

31

37

17

rkpK

LPS* EPS^-

Nod⁺Fix⁺(as wild type)

rkpM

LPS* KPS^-

Pseudonodules, Fix^–

^aLPS* refers to alterations in the electrophoretic profile and lack of recognition by the

monoclonal antibody NB6-228.22.

22

Table 2. Proton and carbon chemical shifts of the OS1_rkpA(A-H, K, Pse^Iand Pse^II) and OS2_rkpA

(E₂, K₂, J, X, and Y) products derived from mild acid hydrolysis and complete deacylation

respectively of LPS from S. fredii HH103 rkpA. 1', 2', 3' and 4' indicate 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^IIwas at 7.51 ppm, whereas that at position 7 of both Pse^Iand Pse^IIwas at 7.18 ppm.

1/1' 2/2'

3/3'

3.69

4/4'

5

6

7

8

9

¹H 5.53 4.03

4.28 4.77

82.5 71.1

4.27 4.81

81.6 71.0

4.19 4.49

70.5 71.9

-

A

4-α-D-GalA ¹³C 99.4 68.3

71.0

175.3

-

¹H 5.50 3.99

3.68

B

4-α-D-GalA ¹³C 99.5 68.4

71.0

175.1

-

¹H 5.09 3.68

3.88

C

t-α-D-GlcA ¹³C 101.2 68.7

69.5

175.5

¹H 5.04 3.53

3.79

4.19 3.91 4.09/3.99

69.5 70.4 66.7

3.38 3.95 3.84/3.64

D

3,6-α-D-Gal ¹³C 99.7 71.6

82.3

¹H 5.00 3.45

3.75

E

t-α-D-Glc

¹³C 97.4 70.3

70.5

70.3 72.9

4.20 4.07

72.1 75.4

3.38 3.19

68.5 76.2

3.31 3.42

68.9 76.0

4.03 3.95

65.5 75.0

3.68 4.05

66.3 48.0

1.13

60.4

-

¹H 4.55 3.92

3.88

F

2-β-D-GalA ¹³C 99.9 76.2

74.0

175.4

3.52

62.2

-

¹H 4.26 3.18

3.62

G

3-β-D-Glc

¹³C 103.5 70.7

81.1

¹H 4.23 3.44

3.40

H

2-β-D-GlcA ¹³C 102.6 81.3

¹H

4,5-α-D-Kdo ¹³C ND 96.2

¹H

3'-β-L,L-Pse ¹³C 172.8 103.2

76.1

174.2

3.69

70.9

3.77

73.0

-

1.94/1.75

33.9

3.87 3.67/3.49

K

69.7

3.94

63.0

4.01

68.3

Pse^I

-

2.42/1.55

36.0

1.09

53.7

16.2

HBu

¹H

¹³C 173.4 45.0

¹H

-

2.24

4.13

69.8

22.0

Pse^II

-

2.47/1.50

36.1

3.77 4.05

66.6 48.0

1.11

3.65

3.86

3.99

1.05

t-β-L,L-Pse ¹³C 172.1 102.3

73.9

53.5

68.8

16.9

HBu

NAc

X

¹H

-

2.22

4.06

¹³C 173.3 44.9

64.8

21.9

1.90

21.8

¹H 5.55 3.30

3.80

69.5

3.71

71.9

3.47 4.06 4.16/3.68

6-α-D-GlcN ¹³C 90.4 54.1

70.1 72.4

3.40 3.67

69.6 75.0

69.2

3.44

61.0

¹H 4.78 2.95

Y

6-β-D-GlcN ¹³C 99.3 55.5

23

¹H 5.08 3.68

3.68

71.9

3.26 4.06

69.8 72.0

4.05 4.20

70.4 72.2

3.83 3.91

69.4 70.1

3.43

61.0

3.62

72.2

3.93

71.4

E₂

2-α-D-Glc

¹³C 97.3 75.4

¹H

-

1.74/1.99

34.3

3.92 3.82/3.55

70.3 62.8

3.87 3.82/3.49

69.6 63.0

K₂

4,5-α-D-Kdo ¹³C ND

ND

-

¹H

¹³C ND

-

1.86/2.11

33.9

J

t-α-D-Kdo

ND

24

Table 3. Macroptilium atropurpureum and Cajanus cajan responses to inoculation with S. fredii

HH103 Rif^R, its rkpM mutant, and its complemented derivative (HH103 rkpM C).

Number of

nodules

Fresh weight of

nodules (mg)

50.53 ± 4.8 A

13.84 ± 2.39 B

48.78 ± 4.68 A

17.53 ± 3.37 B

---

Plant-top dry-

weight (mg)

Legume

Inoculant

HH103 Rif^R

HH103 rkpM

HH103 rkpM C

HH103 rkpA

Uninoculated

39.00 ± 4.77 A

4.33 ± 0.47 B

30.00 ± 2.22 A

8.45 ± 0.81 B

none

127.61 ± 10.03 A

41.43 ± 4.56 B

110.15 ± 11.02 A

63.45 ± 6.23 B

48.83 ± 2.78 B

Macroptilium

atropurpureum

HH103 Rif^R

HH103 rkpM

HH103 rkpM C

Uninoculated

30.58 ± 1.15 A

pseudonodules

31.58 ± 3.17 A

none

273.84 ± 33.84 A

621.22 ± 59.35 A

78.68 ± 5.04 B

722.41 ± 89.13 A

---

353.95 ± 54.42 A

---

Cajanus cajan

101.88 ± 10.72 B

Numbers are mean (± standard error of the mean, SEM) values per plant. M. atropurpureum roots

inoculated with HH103 rkpM also developed pseudonodules. Twelve plants were analysed for each

legume/inoculant combination. For each legume, data in the same column with the same letter were not

significantly different (One-way ANOVA, α = 5%). For “Number of nodules” only those treatments

showing nodules were compared.

analysed

25

Figure legends

Fig. 1. Electrophoretic profile and immuno-detection of the LPS produced by different S. fredii

HH103 Rif^Rmutants affected in LPS and/or KPS production. A) Sodium dodecyl sulphate-

polyacrylamide gel electrophoresis (SDS-PAGE) after silver staining of LPS crude extracts. B)

Immuno-staining using the monoclonal antibody 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 the samples were extracted from free-

living cells, with the only exception of samples HH103 b and rkpM b that correspond to samples

extracted from bacteroids from Glycyrrhiza uralensis nodules induced by HH103 Rif^Rand its

rkpM mutant respectively.

Fig. 2. ¹H NMR spectra of Sinorhizobium fredii HH103 rkpA (top) and rkpM (bottom). Anomeric

signals of spin systems are designated as in Table 2. Diastereotopic methylene proton signals of

Pse^I, Pse^IIand Kdo residues (K/K') are also indicated.

1

Fig. 3. Zoom of the H- ¹³C HSQC spectrum of OS1_rkpAproduct obtained after mild acid

hydrolysis of the LPS from S. fredii HH103 rkpA. Most of the hetero‐nuclear correlations are

indicated. Spin system labels are indicated as in Table 2. The colour of the cross peaks indicates

the phase: black indicates CH and CH₃signals whereas red indicates CH₂signals.

1

Fig. 4. Superimposition of H- ¹³C HSQC (blue/red) with H- ¹³C HMBC (black) of OS1_rkpA

product. Densities are labelled with the same letters listed in Table 2. The most relevant inter-

residue long-range correlations involving sugar moieties are indicated in red, whereas in black

are reported some important intra-residue long-range correlations.

Fig. 5. Structural assessments of the OS1_rkpA, OS2_rkpA, OS1_rkpM,and OS2_rkpMproducts. 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_rkpAand OS1_rkpM) and full deacylation (yielding OS2_rkpAand OS2_rkpM) procedures.

The mild acid hydrolysis selectively cleaved the linkage between the Kdo and the non-reducing

glucosamine of the lipid A portion thus enabling the isolation of the core OS moieties (sugar units

labelled as A, A'-H, H', K, K', Pse^Iand Pse^II) from the lipid A domain. The complete deacylation

procedure allowed the isolation of the core OS plus the glucosamine disaccharide backbone

(labelled as X, X', Y, 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 E₂, E₂'(i.e. the α-D-Glcp substituted at position 2 by the Kdo unit

J, J'). The core OS structures are labelled using letters in Table 2 and S2. “p” indicates the

pyranose form of sugar residues, whereas “P” indicates the phosphate group.

Fig. 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) ad 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.

Fig. 7. A) Immunostaining of intact cells of S. fredii HH103 Rif^Rand HH103 mutants in the rkpA,

rkpM, rkpK, lpsL, lpsB, and lpsE genes using the monoclonal antibody NB6-228.22. LPS: sample

containing HH103 Rif^Rcrude extract preparations used for PAGE electrophoresis (they content

KPS and LPS). KPS: sample containing HH103 lpsB KPS. B) Immunostaining of intact cells of

S. fredii strains isolated from diverse geographical areas and S. meliloti AK631. C)

Immunostaining of LPS electrophoretic patterns of diverse S. fredii strains.

26

Fig. 8. The rkp-3 region organization in the different S. fredii strains analyzed and S. meliloti

Rm41 as reference. Genes in red correspond to the rkpM gene or, in the case of CCBAU45436,

to an orthologue with similar function. The rkp-3 genes shown have different colours depending

on their function.

Fig. 9. Symbiotic performance of HH103 rkpM and its complemented version (HH103 rkpM C)

with Macroptilium atropurpureum (A-C) and Cajanus cajan (D-F). The HH103 rkpA mutant was

also included in the studies with M. atropurpureum. The wild-type strain is denoted as HH103.

NI stands for non-inoculated plants. The phenotype regarding surface polysaccharide production

of mutants rkpM and rkpA is shown is panels A and D: LPS* indicates production of an altered

LPS. A) and D) Aerial part of the plants at the end of the assay. B) and E) Plant leaves. C) and

F) Complete and transversal sections of nodules (or macroscopic root outgrowths) induced by

inoculation with the different strains. The bars in B and E correspond to 1 cm and in C and F to

1 mm.

Fig. 10. Structure of Macroptilium atropurpureum and Cajanus cajan nodules elicited by

inoculation with S. fredii HH103, its rkpM and rkpA mutants, and the rkpM complemented strain

(HH103 rkpM C). M. atropurpureum nodules induced by HH103 Rif^R(A1), HH103 rkpM (A2),

HH103 rkpM complemented (HH103 rkpM C, A3), and HH103 rkpA (A4). C. cajan nodules or

pseudonodules elicited by HH103 Rif^R(B1), HH103 rkpM (B2), and HH103 rkpM C (B3).

Nodules were analysed seven (M. atropurpureum) or eight (C. cajan) weeks after inoculation.

The bar size represents 50 µm.

27

Figures

Fig. 1.

28

Fig. 2

H-2' Pse^I/Pse^II

H-3_ax

H-3_axPse^I

H-3_axPse^II

K

H-3_eqPse^I

H-3_eqPse^II

G

/H

H-3_eq

K

D

B

C

A

F

E

OS1rkpA

H-3_eqK'

H'

G'

H-3_axK'

B'

D'

A'

C'

F'

E'

OS1rkpM

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

ppm

29

Fig. 3

Pse^II9

ppm

20

Pse^I9

NAc

Pse^IPse^II4'

Pse^I3

Pse^II3

Pse^I3

Pse^II3

30

40

50

60

70

80

90

K3

Pse^I/Pse^II2'

Pse^I/Pse^II5

Pse^I7 Pse^II7

E6

Pse^II3'

G6

K8

K4

D6

Pse^I3'

D6

C5

G4

G2

Pse^I4

A5

B5

A2/B2

K5

C4

Pse^II6

F5

F4

F2

G5

B4

D3

A4

G3

H2

E1

A1

D1

C1

B1

F1

H1

G1

100

110

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

ppm

30

Article Doi

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

DOI: 10.1074/jbc.ra120.013393

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.ra120.013393

Full text of DOI:10.1074/jbc.ra120.013393

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