Structure of surface polysaccharides affects symbiotic nitrogen fixation in Frankia alni ACN14a

ABSTRACT Frankia spp. are nitrogen-fixing actinobacteria that establish root-nodule symbiosis with actinorhizal plants comprising more than 200 species in eight dicotyledonous families. From a cell population of Frankia alni strain ACN14a that had been subcultured for over 30 years, we identified two types of variants (types A and S) with a different colony appearance. Type A variants exhibited ambiguous, and type S variants exhibited sharp colony edges. The two types differed in molecular weight and monosaccharide composition of cell surface polysaccharides, which could be responsible for the differences in colony appearance. When inoculated to a host plant Alnus glutinosa, both types induced nodulation but plants infected with type S isolates showed much lower nitrogen fixation activity than those infected with type A isolates. Indeed, type S isolates developed fewer vesicles inside infected plant cells. These results suggest that surface polysaccharides of Frankia play important roles in symbiotic interactions with actinorhizal plants.


Introduction
Frankia is a Gram-positive nitrogen-fixing bacterium that emerged a long time ago and is phylogenetically situated at the root of aerobic actinobacteria (Sen et al. 2014). Frankia establishes root nodule symbiosis with plants collectively called "actinorhizals", which are prevalent in pioneer biotopes such as forest burns, landslides, glacial moraines and manmade mine spoils where they initiate ecological successions (Benson and Silvester 1993). Frankia has been found to belong to more than 13 species and four clades that correlate for the most part with the host spectrum (Normand and Fernandez 2020). The symbiotic determinants of the host plants are now known to be conserved with those of legumes (Griesmann et al. 2018) while those of Frankia remain unknown.
Frankia develops a spherical structure called "vesicle" in which oxygen-labile nitrogenase is expressed. Vesicles are surrounded by a thick hopanoid lipid envelope that functions as a barrier for oxygen, and thus Frankia can fix nitrogen even under aerobic conditions (Parsons et al. 1987). Except for the symbioses with Casuarina and Allocasuarina plants, Frankia also develops vesicles in root nodules. However, morphology of the vesicles differs widely among the actinorhizal plant species, from well-developed spherical structures (Alnus and Elaeagnus) to elongated structures similar to hyphae (Morella) (Silvester et al. 2008).
The outer surface of bacterial cells is associated with various types of polysaccharides which play an important role in host-microbe interactions such as symbiosis and virulence. Rhizobia, which are Gram-negative bacteria establishing rootnodule symbiosis with legumes, have several types of surface polysaccharides (SPs) such as extracellular polysaccharide (EPS), capsular polysaccharide (CPS), lipopolysaccharides (LPS) and cyclic β-glucans (Fraysse et al. 2003;Kucho et al. 2010). EPSs are complex heteropolymers containing multiple repeating units of hexose and they are secreted to the extracellular compartment (Fraysse et al. 2003). Host plants inoculated with rhizobia EPS mutants showed abortion of infection thread elongation and formed ineffective nodules with no or few bacteroids (Leigh et al. 1985;Cheng and Walker 1998;Pellock et al. 2000;Laus et al. 2004;Skorupska et al. 2006). Those nodules exhibited an accumulation of callose, phenolic compounds, phytoalexin, etc., suggesting a role for EPS in the suppression of plant defense responses (Niehaus et al. 1993;Parniske et al. 1994). LPSs consist of three structural parts, O-antigen polysaccharide, core oligosaccharide and lipid A, and they are anchored to the plasma membrane (Fraysse et al. 2003). The majority of rhizobia LPS mutants can invade plant tissues but show reduced nitrogen-fixation activity (Brink et al. 1990;Perotto et al. 1994;Campbell et al. 2002). Mutants lacking O-antigen structures show deficient phenotypes with formation of empty nodules without bacterial colonization (Noel et al. 1986;Stacey et al. 1991).
Among the Gram-positive bacteria, the role of SP in hostmicrobe interaction is well studied in Mycobacterium tuberculosis, the causative agent of tuberculosis. M. tuberculosis has three types of SPs: phosphatidyl-myo-inositol mannosides (PIM), lipomannan (LM) and lipoarabinomannan (LAM) (Flores et al. 2021). Mannan backbone of LAM has arabinan branches, which consist of about 70 arabinofuranose residues. In virulent strains, termini of the arabinan branches have "cap" structures consisting of one to three mannose residues (Mishra et al. 2011). LAM with the "cap" structure causes tuberculosis by inhibiting phagosome maturation and suppressing inflammation (Mishra et al. 2011).
Little is known about the structure and role of SPs in Frankia. Mort et al. (1983) analyzed whole-cell sugar composition in 68 Frankia strains and found that all the strains contained a unique monosaccharide, 2-O-methyl-D-mannose. In the present study, we isolated variants of Frankia alni ACN14a with different SPs properties and investigated their phenotypes in symbiotic nitrogen fixation.

Isolation of the variant colonies
Frankia alni strain ACN14a, which had been subcultured in liquid media for over 30 years, was used for this study. Cells of ACN14a were mutagenized by 1 mg/ml 1-methyl-3-nitro-1-nitrosoguanidine (NTG) for 10 min according to the method described in Kucho et al. (2017). To obtain short hyphae fragments consisting of cells with an identical genotype, hyphae of the strain ACN14a were subjected to the fragmentation and filtration method described previously (Kakoi et al. 2014). Briefly, hyphae were fragmented by ultrasonic treatment and the resulting short fragments were purified by filtration using Ultrafree centrifugal filter units (5 µm pore; Millipore, Billerica, MA, USA). The filtrates were spread onto Frankia defined media (FDM) (Bassi and Benson 2007) supplemented with 0.8 mg/ml proteose peptone No. 3 (BD, Sparks, MD, USA) solidified with 1.2% (wt/vol) agar. The cells were cultivated at 28°C for one month to form colonies. Colonies with different appearances (types A and S, see Results and Discussion) were picked up and propagated in liquid FDM media supplemented with proteose peptone No. 3. We also conducted the same experiment without mutagenesis with NTG.

Preparation and analysis of SP
Cells of Frankia (5 ml of packed cell volume) were disrupted by ultrasonication and then subjected to phenol-water extraction (Fischer 1990) to obtain a crude extract in the aqueous phase. The crude extract was digested with DNase and RNase, and then with proteinase K to obtain crude SPs. The enzymes were removed by dialysis and SPs were recovered by lyophilization.
SDS-PAGE separation of the polysaccharides was performed with the Tris-glycine buffer (Laemmli 1970) using 15% (wt/vol) acrylamide gels. The polysaccharides in the gels were visualized by periodic acid oxidation-silver staining (Tsai and Frasch 1982).
Monosaccharide composition of SPs was analyzed by the alditol acetate method (Torello et al. 1980). Briefly, polysaccharides were hydrolyzed with 2 M trifluoroacetic acid at 100°C for 3 hr, and the hydrolysates were reduced by sodium borohydride followed by acetylation. The alditol acetates were analyzed with a GCMS-QP2010 system (Shimadzu, Kyoto, Japan) equipped with a SP-2330 capillary column (0.25 mm × 30 m, Supelco). The temperature program was initially 150°C for 1 min, followed by an increase to 240°C at a ramp rate of 5°C/min, and a final time migration of 15 min.

Analysis of symbiotic phenotypes
Seeds of Alnus glutinosa were sterilized with 30% (wt/vol) hydrogen peroxide for 30 min and germinated on 0.8% agar plates. Seedlings were transferred to seed pouches (Daiki Rika Kogyo, Saitama, Japan) filled with a nitrogen-free Broughton and Dilworth's solution (Broughton and Dilworth 1971) and grown at 25°C under 16-hr light/8-hr dark regime until length of root reached about 6 cm. Hyphae of Frankia collected from 3 ml cultures were washed twice with sterilized water, resuspended in 3 ml sterilized water, homogenized by repeated passage through 21 G injection needles (Terumo, Tokyo, Japan), and inoculated to seedlings (about 50 μl/plant). Two months after inoculation, nodule number, wet weight of plants and acetylene reduction activity (ARA) were determined. For ARA, we transferred a root system to a sealed test tube. Acetylene was added by injection (5% vol/vol final concentration) and the test tubes were incubated at 25°C for 2 hr. Amounts of ethylene produced were quantified by a gas chromatograph (GC8-AIF, Shimadzu, Kyoto, Japan).
Nodules of A. glutinosa were fixed with a solution containing 5% formaldehyde, 5% acetate and 50% (vol/vol) ethanol and sectioned with a razor blade. The sections were dehydrated with ethanol (30, 50, 70, 80, 90, 95, 100%) and then with tertiary-butyl alcohol. Tertiary-butyl alcohol was removed by sublimation and the sections were observed with the TM-1000S (Hitachi, Tokyo, Japan).

Isolation of variant colonies with different appearance
When we grew cells of Frankia alni strain ACN14a on agar plates, two types of colonies (A and S) with a different appearance were identified. They differed in shape of colony edge (Figure 1). In type A colonies, hyphae radially elongated outward and showed an ambiguous (or fuzzy) edge. In type S colonies, elongation of hyphae was suppressed at the periphery and showed a sharp edge. When edges of colonies were observed with SEM, hyphae were more frequently branched in type S than in type A (Figure 2). About 80% of colonies showed the type A phenotype and the remaining 20% showed the type S phenotype. Mutagenesis with NTG did not affect this ratio, indicating that type S colonies were not induced by the chemical but occurred by spontaneous 1 mm Type A Type S Figure 1. Colonies with different appearance derived from Frankia alni strain ACN14a. Type a colonies showed ambiguous (or fuzzy) edges. Type S colonies showed sharp edges. mutations accumulated over a long time period (>30 years) in these cultures without single-cell isolation. Three representative colonies of type A (A1, A3 and A25) and type S (S4, S9 and S11) were isolated respectively, and used for further experiments. Colonies other than A25 were isolated from a mutagenized cell population. We analyzed nucleotide sequences of 16S rRNA gene of the six isolates and they were identical with the sequence of Frankia alni (data not shown).

Analysis of SP
Alteration in colony appearance is often caused by changes in SP (Güvener and McCarter 2003;Howard et al. 2006;Xu et al. 2008). Therefore, we investigated properties of SPs in the two colony variants. We purified SPs from the six representative isolates and analyzed them with electrophoresis ( Figure 3). The resulting ladder-like pattern was composed of multiple bands ranging from 10 kDa to 30 kDa, indicating that Frankia SP consists of polysaccharide molecules with various sizes. Each of the molecules would contain a different number of repetitive units as reported in other bacteria (Fischer 1990). Bands around 25 to 17 kDa (○ in Figure 3) were relatively thicker in type A isolates than in type S, while bands around 17 to 10 kDa (□ in Figure 3) were thicker in type S than in type A. This result suggests that SP of type S variants is enriched in shorter polysaccharides compared to type A variants.
Monosaccharide composition of the SP was analyzed and we found differences between the two types ( Figure 4). Type S isolates contained more glucose (Glc), mannose (Man), and 2-O-methyl-D-mannose (2-O-Me Man) than type A isolates.
These results indicate that structure of SP is different between the two variant types and this possibly causes the difference in colony appearance. Since 2-O-Me Man is rarely found in SP among microbes and it is a characteristic monosaccharide of Frankia spp. (Mort et al. 1983), the difference was expected to have some effects on the symbiotic interaction.

Phenotypes related to symbiotic nitrogen fixation
The type A and type S isolates were inoculated to a host plant Alnus glutinosa ( Figure 5). Both types induced nodulation although the number of nodules was slightly higher in the type S isolates (Figure 5(a)). However, plants infected with type S isolates showed significantly lower nitrogen fixation activity (ARA) than those infected with type A isolates ( Figure 5(b)). As expected, growth of the plants infected with the type S isolates was much reduced in ammonium-deficient media ( Figure 5(c)) and they developed small yellowish leaves indicative of nitrogen deficiency (Figure 6). Considering the symbiotic phenotypes and the occurrence rate of the colonies (type A 80%, type S 20%) together, type A is wild type and type S is mutant. These results indicate that changes in SP structure affect symbiotic nitrogen fixation of Frankia.

Morphology of Frankia inside nodules
Frankia cells inside nodules were observed by SEM. In nodules induced by type A isolates (A1, A3 and A25), we observed infected plant cells occupied with well-developed Frankia vesicles (Figure 7). In contrast, in the case of type S isolates (S4, S9 and S11), such infected cells were rarely found and Frankia developed fewer vesicles (Figure 7). This result Type A 10 μm Type S 10 μm suggests that defect in nitrogen fixation activity of the type S isolates (Figure 5(b)) was due to a defect in vesicle development.

Possible role of Frankia SP in symbiotic nitrogen fixation
As SPs are displayed on the outer surface of bacterial cells, they can be utilized for recognition of suitable symbiotic partners by host plants. In our case, Frankia isolates with an altered SP structure showed reduction in nitrogen fixation activity ( Figure 5(b)) and vesicle development (Figure 7) in host plant cells. Our result is similar to the cases of LPS mutants of rhizobia, that is, they usually induce nodulation but show reduced nitrogen fixation activity. Mutants of Rhizobium leguminosarum with altered monosaccharide composition in LPS induced incompletely developed clover nodules exhibiting low bacterial population and low nitrogenase activity (Brink et al. 1990). R. leguminosarum mutants lacking high-molecular-weight LPS exhibited severely reduced nitrogen fixation activity in pea nodules, and the host plant cells exhibited defense responses characterized by callose deposition and cell death (Perotto et al. 1994). Furthermore, alfalfa nodules formed by a LPS mutant of Sinorhizobium meliloti contained abnormally developed bacteroids, and exhibited reduced nitrogen fixation ability (Campbell et al. 2002). The S. meliloti LPS mutant was sensitive to antimicrobial peptides that are components of plant's innate immune system. Nodules of Alnus gultinosa also contain antimicrobial peptides that control activity of Frankia in infected cells (Carro et al. 2015(Carro et al. , 2016. The difference in SP structure revealed in our study may have altered sensitivity of Frankia cells to the antimicrobial peptides and caused the difference in nitrogen-fixing ability between types A and S variants. Alternatively, it is also possible that the difference in SP structure affected recognition of Frankia cells by host plants because the content of 2-O-Me Man, which is a sugar characteristic of Frankia (Mort et al. 1983), was different between the two variants.

A1
A3 A25 S4 S9 S11 Not inoculated The genome of F. alni strain ACN14a contains four SP synthesis gene clusters (Table S1). Each cluster is composed of 12-35 genes and contains 9-19 genes with predicted functions related to SP biosynthesis, such as nucleotide sugar precursor biosynthesis (N in Table S1), sugar transfer (T) and processing of sugar chains (P) (Samuel and Reeves 2003). Since the content of Glc, Man, 2-O-Me Man in SPs were different between the type A and the type S isolates (Figure 4), genes related to metabolism of those sugars -FRAAL1228 (dTDP-glucose synthase), FRAAL2038 (putative O-methyltransferase), FRAAL2056 (GDP-D-mannose dehydratase), FRAAL2058 (putative UDP-glucose/GDP-mannose dehydrogenase), FRAAL6241 (CDP-glucose pyrophosphorylase), FRAAL6248 (putative O-methyltransferase), and FRAAL6256 (UDPglucose epimerase/dehydratase) -may have genetic variations between the two types. In addition, expression of the two genes -FRAAL1239 (putative polysaccharide ABC transport integral membrane subunit) and FRAAL1248 (putative glycosyltransferase) -were slightly upregulated in A. glutinosa nodules (Alloisio et al. 2010), suggesting that these genes could have a role with the symbiotic phenotypes observed in this study.
In conclusion, we isolated two Frankia alni variants with different colony appearance and found that they differed in the structure of their SP. The type S variants showed less nitrogen fixation activity and impaired vesicle development in Alnus glutinosa nodules compared to the type A variants. Comparison of genome sequences between the two variants should help identify the gene responsible for the phenotypes.