*3.2. Lectin Binding Profiles of Bradyrhizobium Strains Change When Cultured in SESOM*

The presence of surface-exposed sugars was characterized semi-quantitatively using 24 lectins. Lectin binding intensity was scored by visible fluorescence after different exposure times, yielding a lectin fingerprint for each strain (Figure 3). USDA 110 bound to the highest number of lectins, followed by strains 26, 126 and then 3384. Each strain bound not one but multiple lectins, but also displayed binding to a unique combination of lectins, indicating different combinations of surface-exposed sugars. The fluorescence intensity varied among lectins and strains, indicating differences in the quantity of bound lectin. Importantly, SESOM-grown populations all bound fewer lectins than those grown in PSY. This indicates a decrease in the diversity of surface-exposed sugars and therefore shifts in extracellularly expressed polysaccharides. SESOM-grown USDA 110 bound only four of the 13 lectins bound ex PSY. Intriguingly, no new lectin binding was observed ex SESOM, except for USDA 126 which bound sWGA and STL. Collectively, this indicates a decrease in the variety of surface-exposed sugars in *Bradyrhizobium* when growing in a soil environment.

**Figure 3.** Lectin binding profiles of *Bradyrhizobium* strains cultured in either PSY-Arabinose or SESOM to mid-exponential phase. Binding intensity was scored by the shortest exposure time yielding visible fluorescence, with 100 ms scoring level 4, and <1200 ms scoring level 0.

Only a small proportion of cells displayed fluorescence, so cells appeared to either bind or not bind a specific lectin (Figure 4). To ascertain whether this was due to an insufficient quantity of lectin in the binding reaction, cultures were exposed to increased lectin concentrations, up to 300 μg/mL. This did not increase the proportion of lectin binding cells (data not shown), but instead indicated the phenotypic variability among exponential phase cells in a liquid culture. As the slow-growing *Bradyrhizobium* are more prone to contamination during culture, experimentation included regular sub-culturing on agar to ensure cultures were not contaminated. SESOM-cultured populations generally displayed lower proportions of lectin binding cells than ex PSY, with some exceptions such as USDA 110 binding to WGA and USDA 126 binding to RCA I. These results indicate a differentiation in surface properties among cells in *Bradyrhizobium* populations cultured under homogenous conditions (liquid shake culture).

**Figure 4.** Lectin binds only to a subset of cells in the population. Binding of sWGA to *B diazoefficiens* USDA 26 grown in SESOM (**A**) and PSY (**B**), and the percentage of cells binding to the 25 lectins after culturing in PSY and SESOM (**C**).

## *3.3. SESOM-Grown Populations Form No Biofilm but Display Increased Adherence to Soy Roots*

*Bradyrhizobium* growing in SESOM displayed altered attachment-associated traits, so we quantified in vitro biofilm formation using the standard microtitre plate biofilm assay. PSY-grown USDA 110 and 26 formed biofilms on polystyrene within 24 h (Figure 5A), while USDA 126 and 3384 did not, even when incubated for 96 h (data not shown). Intriguingly, no strain formed biofilm in SESOM, with cells remaining in the planktonic phase. These results indicate that *Bradyrhizobium* growing in soil would not attach and form biofilms on more hydrophilic surfaces such as the Nunclon Delta Surface Polystyrene used in these experiments.

**Figure 5.** Biofilm (**A**) and soy root adherence (**B**) of *Bradyrhizobium* strains. Exponential phase *Bradyrhizobium* cultured in either PSY-Arabinose or SESOM were inoculated into 96-well polystyrene plates and biofilm formed after 24 h quantified by crystal violet staining (**A**), or exposed to 9 d old roots for 60 min (**B**). Data were analyzed by Student *t*-test, and *p* values indicate statistical significance (ns not significant, \*\*\*\* *p* < 0.0001).

To gain insight into association with young soy roots, *Bradyrhizobium* was grown in PSY or SESOM, suspensions of exponentially growing cells were exposed to 9-day-old gnotobiotic seedlings for 60 min, and the adhering cells were counted. Adherence of all four PSY-cultured populations was scant (Figure 5B). In contrast, SESOM-grown USDA 110 displayed significantly more root adhesion. The adhesion data obtained for SESOM-grown cultures were more varied than for ex PSY. While root adhesion of strains 26, 126 and 3384 was not found statistically significant, it was higher than in PSY in multiple cases (Figure 5B). This greater variability in observed phenotype among individual experiments may be due to either variability among young roots, or due to variation in *Bradyrhizobium* population phenotype, as is also observed in lectin binding experiments.

## **4. Discussion**

Our data reveal that *Bradyrhizobium* adopts adhesion-related surface properties when grown in the liquid soil extract SESOM. We observed the effect of soil nutrient environment on the surface hydrophobicity, motility, lectin binding spectrum and adherence capacity of the cells to soy roots. SESOM-grown populations of strains 110 and 3384 were more hydrophobic in nature, whereas there was no significant difference with strains 26 and 126. An effect of growth media on the surface hydrophobicity of bacterial cells has been reported for other bacterial species [25–27]. The hydrophobic properties of both the root surface and the bacteria involved in symbiosis contributes to one of the strongest forces in the initial root association. Bacterial cell surface hydrophobicity has been correlated directly to root attachment and competitive nodule forming ability [6,28]. Structural changes brought in LPS increase the cell surface hydrophobicity, which likely is significantly involved in the endocytotic infection process [7]. It is likely that the soil nutrients make 110 and 3384 more hydrophobic, and hence increase their chance of attachment by supporting them in their competitiveness to other bacteria. This was observed in the root adherence assay with strain 110 but not 3384.

The motility of rhizobia in response to chemical signaling plays a critical role in the initial contact, attachment and colonization of the root hair [29]. Mutants of *Rhizobium meliloti LS-30* with defective motility took 5–20 times more time to adsorb to the root cell, and had delayed nodule formation, making them less competitive than other bacteria [11]. In this study, SESOM-grown populations were observed to be less motile. This could be because *B. japonicum* grown in L-arabinose as sugar source expresses lateral flagella along with subpolar flagella, making them move faster than those grown in other sugars [17]. Our lectin data indicated reduced surface sugar diversity when growing in SESOM. Alterations of the surface sugar moieties and their effect on symbiotic capability have been observed in *Bradyrhizobium* [9,30–32]. Surface sugar diversity has been reported to vary in composition when grown on different carbon sources [33,34]. These data suggest that the carbon sources in SESOM lead *Bradyrhizobium* to produce a different combination of extracellular polysaccharides and other sugar-containing polymers. Specifically, SESOM-grown populations appear to produce a smaller number of such extracellular polymers.

The contrast between biofilm formation on polystyrene and adherence to soy roots was unexpected, especially in the case of strain 110. While SESOM-grown populations failed to form biofilm on polystyrene, they were more prone to adhere to the soy roots. Arabinose as carbon source yielded opposite outcome. Medium characteristics such as osmolarity, nutrients, and factors derived from the biotic environment, may also affect attachment and/or biofilm formation. Little is known about the distribution of surface properties on young soybean roots, but the SESOM-grown, more hydrophobic strain 110 attached to the roots. In contrast it did not form detectable biofilm on the hydrophilic-modified Nunclon Delta Surface plates used. As confirmation, the hydrophilic ex PSY populations attached to the hydrophilic polystyrene, but not the soy roots. Biofilm formation of *Rhizobium* is affected by growth medium and incubation conditions [35]. Soybean agglutinin (SBA) lectin is thought to contribute to root-surface adherence [36]. While strains 110 and 126 displayed SBA binding under both culture condition (Figure 3), root adherence was significantly different. This suggest that root adherence may be driven largely by other factors.

The binding of specific lectins to only some cells in the population was surprising. Selective binding was not due to a shortage of lectin concentration as confirmed by experiments with increased concentration. The purity of the culture had also been verified regularly, so selective lectin binding pointed to differentiation in surface-exposed sugar polymers. This apparent differentiation of surface properties of cells in liquid shake cultures was unexpected. Our methodology included the removal of residual sugars from the culture medium by washing cells, and a wash step to remove unbound lectins after binding. These wash steps could have contributed to a loss of EPS into the liquid phase, leading to less or no bound lectin retained on cells. A similar observation of differential polysaccharide positioning has also been reported in *Agrobacterium tumefaciens* and

*Rhizobium leguminosarum* [37,38]. While phenotypic population differentiation has been studied extensively in *Bacillus subtilis* [39,40], it is better known from environments allowing niche-specific physicochemical conditions such as in biofilms. The ecophysiological role of phenotypic differentiation of *Bradyrhizobia* presents itself as a new field of study.

From the results, it is clear that growth in a soil nutrient environment does affect the surface properties and adherence capacity of *Bradyrhizobium.* This is true especially for USDA 110, which is an agriculturally important rhizobial bacteria for soybean nitrogen fixation, where SESOM increased hydrophobicity and root adherence capability significantly [41]. The observed differences in the surface and phenotypic properties of the soil nutrient grown population leads to the conclusion that *Bradyrhizobium* adjust with the chemical cues that are present around them. The results from this study emphasize the need to further explore the effect of (secreted) root exudates on the surface and phenotypic behavior of *Bradyrhizobium* cells and resulting root adherence.

**Author Contributions:** Conceptualization, S.S. and V.S.B.; methodology, A.K.S. and V.S.B.; formal analysis, A.K.S.; investigation, A.K.S.; resources, S.S.; data curation, A.K.S.; writing—original draft preparation, A.K.S.; writing—review and editing, A.K.S., S.S. and V.S.B.; visualization, A.K.S.; supervision, V.S.B.; project administration, S.S; funding acquisition, S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This material is based upon work supported by the National Science Foundation/EPSCoR RII Track-1: Building on The 2020 Vision: Expanding Research, Education and Innovation in South Dakota, Award OIA-1849206 and by the South Dakota Board of Regents.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We thank the NRRL Culture Collection of the Agricultural Research Service, United State Department of Agriculture for providing the bacterial cultures used in this study. We acknowledge use of the SDSU-FGCF supported in part by NSF/EPSCoR Grant No. 0091948 and by the State of South Dakota. We thankKMTaufiqur Rahman for his help with image analysis.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Disclaimer:** Any opinions: findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

## **References**


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