Surface Motility Regulation of Sinorhizobium fredii HH103 by Plant Flavonoids and the NodD1, TtsI, NolR, and MucR1 Symbiotic Bacterial Regulators

Citation: Alías-Villegas, C.; Fuentes-Romero, F.; Cuéllar, V.; Navarro-Gómez, P.; Soto, M.J.; Vinardell, J.-M.; Acosta-Jurado, S. Surface Motility Regulation of Sinorhizobium fredii HH103 by Plant Flavonoids and the NodD1, TtsI, NolR, and MucR1 Symbiotic Bacterial Regulators. Int. J. Mol. Sci. 2022,23, 7698. https://doi.org/10.3390/ ijms23147698 Academic Editor: Alessio Mengoni Received: 23 June 2022 Accepted: 11 July 2022 Published: 12 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Surface Motility Regulation of Sinorhizobium fredii HH103 by Plant Flavonoids and the NodD1, TtsI, NolR, and MucR1 Symbiotic Bacterial Regulators Cynthia Alías-Villegas 1, Francisco Fuentes-Romero 2, Virginia Cuéllar 3, Pilar Navarro-Gómez 2, María J. Soto 3, José-María Vinardell 2,* and Sebastián Acosta-Jurado 1,* 1Centro Andaluz de Biología del Desarrollo, CSIC/Junta de Andalucía, Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, 41013 Seville, Spain; [email protected] 2Facultad de Biología, Departamento de Microbiología, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (F.F.-R.); [email protected] (P.N.-G.) 3Estación Experimental del Zaidín, CSIC, Departamento de Biotecnología y Protección Ambiental, 18008 Granada, Spain; [email protected] (V.C.); [email protected] (M.J.S.) *Correspondence: [email protected] (J.-M.V.); [email protected] (S.A.-J.); Tel.: +34-954-554-330 (J.-M.V.); +34-954-348-565 (S.A.-J.) Abstract: Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α - and β -Proteobacteria, establish nitrogen-fixing symbioses with legumes that rely on a complex signal interchange between the partners. Flavonoids exuded by plant roots and the bacterial transcriptional activator NodD control the transcription of different rhizobial genes (the so-called nod regulon) and, together with additional bacterial regulatory proteins (such as TtsI, MucR or NolR), influence the production of different rhizobial molecular signals. In Sinorhizobium fredii HH103, flavonoids and NodD have a negative effect on exopolysaccharide production and biofilm production. Since biofilm formation and motility are often inversely regulated, we have analysed whether flavonoids may influence the translocation of S. fredii HH103 on surfaces. We show that the presence of nod gene-inducing flavonoids does not affect swimming but promotes a mode of surface translocation, which involves both flagella-dependent and -independent mechanisms. This surface motility is regulated in a flavonoid-NodD1-TtsI-dependent manner, relies on the assembly of the symbiotic type 3 secretion system (T3SS), and involves the participation of additional modulators of the nod regulon (NolR and MucR1). To our knowledge, this is the first evidence indicating the participation of T3SS in surface motility in a plant-interacting bacterium. Interestingly, flavonoids acting as nod-gene inducers also participate in the inverse regulation of surface motility and biofilm formation, which could contribute to a more efficient plant colonisation. Keywords: Sinorhizobium fredii; surface motility; swarming; swimming; NodD; TtsI; NolR; slavonoids; T3SS; flagella 1. Introduction Rhizobia are soil bacteria able to establish a mutualistic relationship with plants belonging to the Fabaceae family. In this interaction, bacteria infect legume roots and colonise new organs, called nodules, formed by the plant. Inside nodules, rhizobia invade plant cells and differentiate into bacteroids able to fix atmospheric N 2 into ammonia that is provided to the plant. In return, the plant supplies to the bacteria a carbon and energy source as well as an appropriate environment for nitrogen fixation [1,2]. The rhizobia–legume symbiotic interaction requires a coordinated, continuous and complex interchange of molecular signals between compatible rhizobia and legumes [ 3 – 6 ]. The nodulation process is initiated by the root exudation of a plethora of molecules which Int. J. Mol. Sci. 2022,23, 7698. https://doi.org/10.3390/ijms23147698 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 7698 2 of 18 includes phenolic compounds called flavonoids [ 7 ]. The bacterial transcriptional regulator NodD interacts with appropriate flavonoids and, through its binding to conserved promoter sequences named nod boxes (NB) [ 8 , 9 ], activates the transcription of genes responsible for the production and secretion of bacterial molecular signals called Nod factors (NF, or lipochitooligosaccharides, LCOs). These NF are N-Acetyl-glucosamine oligosaccharides harbouring different molecular decorations and, when appropriate, are recognised by LysM receptors located on the root hair cells, triggering root infection and the formation of nodule primordia in the root cortex. In most legumes, bacteria reach the symbiotic cortical cells of nodules through tubular structures, called infection threads, that initiate in root hairs and connect the epidermal and the cortical layers of the root [ 3 , 10 , 11 ], although there are also intercellular infection strategies, such as crack entry [12]. Besides NF, other bacterial molecular signals may be important for an efficient symbiosis [4,13] . In some rhizobia, such as the broad host range Sinorhizobium fredii HH103, NodD also induces the expression and assembly of a type 3 secretion system (T3SS) through the activation of the expression of the ttsI transcriptional activator. The T3SS delivers effector proteins into host plant cells and modifies plant defence responses [ 5 , 14 , 15 ]. Rhizobial surface polysaccharides (RSP) can also act as signal molecules during the symbiotic interaction and/or as a protection against plant defences. Diverse RSP have been described in rhizobia, such as exopolysaccharides (EPS), the K-antigen capsular polysaccharide (KPS), lipopolysaccharides (LPS) and cyclic glucans (CGs) [ 4 , 13 ]. In S. fredii HH103, in addition to NodD1 (the master positive regulator of nod genes) and TtsI, other rhizobial transcriptional regulators, such as NolR (a global repressor of the nod regulon), MucR (another global regulator involved in multiple processes in both saprophytic and symbiotic stages), SyrM and NodD2 (both acting as activators and repressors of different genes of the nod regulon) play important roles in the fine tuning of the production of bacterial molecular signals involved in symbiosis [16–21]. In nature, microorganisms usually live within biofilms, complex microbial communities assembled in an extracellular matrix (containing bacterial EPS, as a key component) that adhere to a surface [ 22 ]. In their transition from soil saprophytes to legume endosymbionts, rhizobia attach and form biofilms on roots and root hairs prior to infecting legume roots [ 2 ]. Thus, the production of EPS is an important trait for colonisation of legume root surfaces by rhizobia. In fact, flavonoids exuded by legume roots have a positive effect on R. leguminosarum bv. trifolii EPS production [ 23 ]. However, in S. fredii HH103 nod gene-inducing flavonoids repress EPS production and biofilm formation in a NodD1- and SyrM-dependent manner [21,24], whereas NolR stimulates both processes [16]. Biofilm formation and surface motility are opposite behaviours exhibited by bacteria living on surfaces [ 22 , 25 ]. Rhizobial motility is not essential for nodulation and nitrogen fixation but can influence nodulation efficiency and competitiveness by approaching bacteria to potential infection sites and/or facilitating their progression inside infection threads [ 26 – 28 ]. Several studies have shown that rhizobial mutants affected in flagella synthesis exhibit partial defects in symbiotic performance such as reduced ability for root colonisation [ 29 ], delayed nodulation and/or reduced competitiveness for nodulating host legumes [ 25 , 30 – 33 ]. These results suggest that rhizobial motility may be an important trait for host root colonisation and infection. In fact, since rhizobia lack flagella inside infection threads, Fournier et al. [ 26 ] proposed that rhizobial penetration through these tubular structures is the result of the combination of surface translocation (flagella-independent) and cell division. On the other hand, in some studies genes related to flagellar biosynthesis have been shown to be strongly repressed inside nodules [ 34 – 36 ], in agreement with the non-motile state of bacteroids (the rhizobial differentiate forms present inside nodule cells and able to fix nitrogen). However, a recent work has shown that most flagellar assembly and basal body apparatus proteins are important in R. leguminosarum bacteroids and nodule bacteria in symbiosis with Pisum sativum [27]. Rhizobia can move using different mechanisms. The most studied is swimming, a flagella-driven motility that allows the movement of individual bacteria in liquid media. Int. J. Mol. Sci. 2022,23, 7698 3 of 18 Nevertheless, like many other bacteria, rhizobia can also move on surfaces using flagelladependent and independent mechanisms such as swarming, surfing or sliding [ 37 – 41 ]. Swarming and surfing are flagella-driven motilities, whereas sliding is a passive appendageindependent bacterial translocation. Swarming is the fastest known type of bacterial motility on surfaces, and it is characterized by the coordinated multicellular migration of cells. This type of motility has been described in several rhizobial species [ 29 , 33 , 42 – 47 ]. In contrast to swarming, surfing and sliding have only been described in the alfalfa symbiont Sinorhizobium meliloti [ 33 , 45 , 48 ]. Surface spreading in surfing is driven by flagellar rotation and physical/chemical effects created by a secreted exopolysaccharide (EPS), whereas sliding bacterial translocation is powered by the expansive forces of cell growth and facilitated by the production of surfactants that reduce surface tension. Bacterial translocation on surfaces is greatly influenced by numerous environmental factors, which makes it a complex behaviour to study [ 40 ]. In rhizobia, knowledge about the regulatory mechanisms and environmental cues that control surface motility is still limited. Specifically, studies that investigate the influence of root exudates on the ability of rhizobia to spread on surfaces are scarce [ 43 , 44 , 49 ]. Noteworthy, it has been proposed that the presence of strigolactones (phytohormones that regulate different developmental processes) in plant root exudates promote bacterial surface translocation in some rhizobial species, such as S. meliloti GR4 and Rhizobium leguminosarum bv. viciae [50,51]. In several soil bacteria, biofilm formation and surface motility are inversely regulated [22,25]. Since nod gene-inducing flavonoids and NodD1 negatively affect EPS production and biofilm formation in S. fredii HH103, in this work we wanted to investigate whether the presence of these molecules also had an effect on S. fredii motility. Our data show that the presence of nod gene-inducing flavonoids such as genistein stimulates surface translocation in S. fredii HH103 but does not affect swimming. The genistein-induced surface motility, which is mediated by both flagella-dependent and -independent mechanisms, is dependent on NodD1, TtsI, and the presence of a functional T3SS. We also show that additional modulators of the nod regulon, NolR and MucR, participate in the control of HH103 flavonoid-induced surface motility. 2. Results 2.1. nod Gene-Inducing Flavonoids Promote Surface Motility in S. fredii HH103 In previous reports, S. fredii HH103 showed a slight surface translocation on 0.6% agar semisolid minimal medium (MM) that was promoted by EPS production [ 52 ]. Since the presence of nod gene-inducing flavonoids reduces EPS production and biofilm formation by HH103 [ 24 ], we decided to study whether these compounds also had an effect on HH103 surface motility. In these new experiments, agarose at 0.4% (w/v) was used as the gelling agent because it increased the reproducibility. Surprisingly, the presence of genistein, an effective inducer flavonoid of HH103 nod genes present in soybean root exudates [ 53 ], provoked a significant increase in the HH103 surface propagation capacity (76.6 ± 6.6 mm) in comparison to that exhibited in the absence of added compounds ( 8.2 ±3.1 mm ) on semisolid MM 0.4% agarose after 72 h (Figure 1). In order to investigate whether this induction was specific for genistein or was also promoted by other nod gene-inducing flavonoids, we carried out surface motility experiments in the presence of two other effective inducers of HH103, coumestrol and apigenin, and three flavonoids unable to induce nod gene expression in this strain: flavanone, 6-hydroxyflavanone, and 5-hydroxyflavone [ 53 ]. The results obtained showed a significant increase in HH103 surface translocation in the presence of either coumestrol or apigenin, 64.1 ± 8.4 mm and 68.5 ±6.4 mm , respectively, although these increases were significantly lower than that obtained in the presence of genistein. In contrast, the surface spreading areas obtained in the presence of non-inducing flavonoids were indistinguishable from those observed under control conditions (ethanol; 8.2 ± 4.7 mm) or in the absence of any added compound to MM (8.2 ± 3.1 mm): 10.5 ± 6.4 mm for flavonone, 8.8 ± 5.2 mm for 6-hydroxyflavanone, and 9.8 ±5.7 for 5-hydroxyflavone (Figure 1). Int. J. Mol. Sci. 2022,23, 7698 4 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 18 conditions (ethanol; 8.2 ± 4.7 mm) or in the absence of any added compound to MM (8.2 ± 3.1 mm): 10.5 ± 6.4 mm for flavonone, 8.8 ± 5.2 mm for 6-hydroxyflavanone, and 9.8 ± 5.7 for 5-hydroxyflavone (Figure 1). Figure 1. Effect of different compounds (including nod gene-inducing and non-inducing flavonoids) on S. fredii HH103 surface motility. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as the gelling agent and scored at 72 h. (A). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. (B). Representative pictures of the motilities exhibited by S. fredii HH103 under each condition. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). 2.2. The HH103 Genistein-Induced Surface Motility Is Mediated by Flagella-Dependent and - Independent Mechanisms Bacterial surface motility can be mediated by flagella-dependent or flagella-inde- pendent mechanisms [38,41]. In order to elucidate whether the genistein-induced surface spreading exhibited by S. fredii HH103 is a flagella-dependent movement, we carried out Figure 1. Effect of different compounds (including nod gene-inducing and non-inducing flavonoids) on S. fredii HH103 surface motility. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as the gelling agent and scored at 72 h. ( A ). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney , α = 1%. ( B ). Representative pictures of the motilities exhibited by S. fredii HH103 under each condition. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). 2.2. The HH103 Genistein-Induced Surface Motility Is Mediated by Flagella-Dependent and -Independent Mechanisms Bacterial surface motility can be mediated by flagella-dependent or flagella-independent mechanisms [ 38 , 41 ]. In order to elucidate whether the genistein-induced surface spreading exhibited by S. fredii HH103 is a flagella-dependent movement, we carried out surface motility experiments with a flagella-less derivative, in which the flaCBAD genes were deleted [ 52 ]. The results showed that the surface propagation in the presence of the genistein of the ∆ flaCBAD derivative (45.4 ± 7.8 mm) was reduced by almost 40% when compared to the wild type, indicating that the S. fredii HH103 genistein-induced surface Int. J. Mol. Sci. 2022,23, 7698 5 of 18 motility is the result of a combination of both flagelladependent and independent surface motilities (Figure 2). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 18 surface motility experiments with a flagella-less derivative, in which the flaCBAD genes were deleted [52]. The results showed that the surface propagation in the presence of the genistein of the ΔflaCBAD derivative (45.4 ± 7.8 mm) was reduced by almost 40% when compared to the wild type, indicating that the S. fredii HH103 genistein-induced surface motility is the result of a combination of both flagelladependent and independent surface motilities (Figure 2). Figure 2. Role of different symbiotic regulators on the S. fredii HH103 surface translocation in the presence of genistein. The non-flagellated (flaCBAD) and nodA mutants were also included. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as gelling agent and scored at 72 h. (A). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. (B). Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). Figure 2. Role of different symbiotic regulators on the S. fredii HH103 surface translocation in the presence of genistein. The non-flagellated (flaCBAD) and nodA mutants were also included. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as gelling agent and scored at 72 h. ( A ). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. ( B ). Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). 2.3. The HH103 Genistein-Induced Surface Motility Depends on the Regulators NodD1, TtsI and NolR Since the S. fredii HH103 surface motility was specifically promoted by nod geneinducing flavonoids, we decided to investigate whether the regulatory proteins involved in the control of nod gene expression had a role in this motility. For this purpose, we Int. J. Mol. Sci. 2022,23, 7698 6 of 18 analysed the surface propagation in response to genistein of HH103 mutants affected in different symbiotic regulators: NodD1, the activator of the nod regulon in response to the presence of flavonoids; TtsI, which is the transcriptional activator of the symbiotic T3SS; and different regulatory proteins that take part in the fine-tuning of the nod regulon: NodD2, SyrM, NolR and MucR1 [ 16 , 17 , 19 – 21 , 53 ]. More specifically, the HH103 mutants examined were: nodD1:: Ω [ 53 ], nolR::lacZ ∆ p-Gm R [ 24 ], nodD2:: Ω [ 20 ], ∆ mucR1 [ 19 ], ∆ syrM [ 21 ], and a HH103 derivative in which the Ω interposon was inserted into the nod box that regulates the genistein-induced expression of ttsI (indicated as PttsI) [ 17 ]. In addition, in order to check whether NF could be involved in the genistein-induced surface motility, we included anodA mutant unable to produce these signal molecules [54]. In the absence of flavonoids, and similarly to the wild-type strain, none of the mutants tested exhibited surface motility (Figure 2). However, the results obtained suggested that the genistein-induced surface spreading exhibited by S. fredii HH103 is dependent on the NodD1, TtsI and NolR regulatory proteins, since the lack of any of them provoked a severe reduction in genistein-induced surface motility or a complete abolition in the case of the nolR mutant (nodD1 19.5 ± 9.5 mm; ttsI 19.4 ± 5.3 mm; nolR 6.6 ± 1.1 mm; Figure 2) when compared to the wild-type strain (78.8 ± 3.8 mm). In contrast, the HH103 nodD2 and syrM mutants did not show significant differences in their propagation levels in comparison to the wild type (79.4 ± 6.8 mm and 74.8 ± 9.6 mm, respectively, Figure 2). An intermediate degree of genistein-induced surface translocation was observed in the mucR1 mutant (37.6 ±9.8 mm) . The nodA mutant exhibited a slight but significant reduction ( 66.8 ±12.6 mm ;p= 0.05) of this motility, suggesting that NF can influence HH103 surface propagation ability. In order to discard that the impaired surface motility exhibited by some of the mutants could be caused by growth defects on the medium employed for the surface motility experiments, growth curves of the wild-type strain and the different mutants were performed in liquid MM. As shown in Figure S1, none of the mutants assayed exhibited significant differences in growth when compared to the wild-type strain. The participation of NodD1, NolR, MucR, and TtsI in the genistein-induced surface translocation exhibited by HH103 was confirmed by analysing the behaviour of additional independent mutants in the corresponding regulatory genes: nodD1::lacZ ∆ p-Gm R [ 55 ], nolR:: Ω [ 17 ], mucR1::lacZ ∆ p-Gm R [ 19 ], and ttsI:: Ω [ 17 ]. The genistein-induced surface spreading ability of all these mutants (Figure S2) was similar to that of the previously analysed mutants and, therefore, significantly reduced when compared to that of the wildtype strain: 11.2 ± 0.8 for nodD1::lacZ ∆ p-Gm R , 6.3 ± 1.8 mm for nolR:: Ω , 40.8 ± 9.0 for mucR1::lacZ∆p-GmR, and 12.6 ±4.1 mm for ttsI:.Ω. 2.4. S. fredii HH103 Swimming Motility Is Not Affected by Genistein Once we confirmed the positive effect of genistein on the ability of S. fredii HH103 to move over surfaces, we wanted to investigate whether this inducer flavonoid could also affect another type of motility, swimming. For this purpose, motility experiments on semisolid BM 0.3% agar in the presence of either genistein or ethanol were performed. As shown in Figure 3, the swimming motility exhibited by HH103 at 72 h upon genistein or ethanol treatment were similar, 14.2 ± 1.2 mm and 14.5 ± 3.6 mm, respectively. In relation to this result, we checked whether any of the HH103 mutants affected in different symbiotic regulators might show changes in swimming motility behaviour in both conditions. The S. fredii HH103 ∆ flaCBAD derivative was also included as a non-motile control. Except for the latter strain (4.5 ± 0.5 mm), no significant differences in the swimming rings were obtained among the different mutants tested in comparison to those of the wild type in the presence of both genistein and ethanol (Figure 3). Int. J. Mol. Sci. 2022,23, 7698 7 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 18 obtained among the different mutants tested in comparison to those of the wild type in the presence of both genistein and ethanol (Figure 3). Figure 3. Swimming abilities of S. fredii HH103 and different mutants in the absence and the presence of genistein at 72 h. All data points are shown in box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed are also shown. Values under images represent the average and standard deviation of migration (given in millimetres and determined as described in the text). Since genistein and different symbiotic regulatory proteins influence the surface motility of S. fredii HH103, we also investigated whether they affect flagella production in this bacterium by using transmission electron microscopy. The samples for TEM studies were taken from surface motility assays at 24 h in the presence of either genistein or ethanol. The HH103 ΔflaCBAD mutant was used as a negative control of the presence of flagella. The results obtained revealed that the presence or absence of genistein apparently does not affect flagellum production by S. fredii HH103 (Figure 4). All the mutants tested showed flagella and no clear differences could be scored when compared to the wild-type strain (Figure 4). These results, together with the swimming motility data, suggest that neither genistein nor the lack of NodD1, NolR or TtsI have a clear effect on flagellum production. Figure 3. Swimming abilities of S. fredii HH103 and different mutants in the absence and the presence of genistein at 72 h. All data points are shown in box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed are also shown. Values under images represent the average and standard deviation of migration (given in millimetres and determined as described in the text). Since genistein and different symbiotic regulatory proteins influence the surface motility of S. fredii HH103, we also investigated whether they affect flagella production in this bacterium by using transmission electron microscopy. The samples for TEM studies were taken from surface motility assays at 24 h in the presence of either genistein or ethanol. The HH103 ∆ flaCBAD mutant was used as a negative control of the presence of flagella. The results obtained revealed that the presence or absence of genistein apparently does not affect flagellum production by S. fredii HH103 (Figure 4). All the mutants tested showed flagella and no clear differences could be scored when compared to the wild-type strain (Figure 4). These results, together with the swimming motility data, suggest that neither genistein nor the lack of NodD1, NolR or TtsI have a clear effect on flagellum production. Int. J. Mol. Sci. 2022,23, 7698 8 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 18 Figure 4. Transmission electron microscope (TEM) images of S. fredii HH103 and different mutants. Cells were isolated from the edge of colonies grown on semisolid MM 0.4% agarose in the presence of genistein or ethanol after 24 h of incubation at 28 °C and stained with 2% uranyl acetate. Scale bars correspond to 1 µm. 2.5. A Functional T3SS Is Required for the S. fredii HH103 Genistein-Induced Surface Motility As mentioned above, two independent mutants affected in the ttsI gene exhibited a dramatic reduction in the genistein-induced surface motility of S. fredii HH103, suggesting a role of the HH103 T3SS in this kind of motility. Because of this, we tested the surface motility of HH103 mutants affected in different genes that code for structural components of the S. fredii HH103 T3SS: RhcJ (=NolT), an inner MS ring protein; RhcV (=NolV), a structural protein anchored to the inner membrane; and NopA, the T3SS pilus subunit [14,17]. The mutants analysed were rhcJ::Tn5-lacZ [56], rhcV::Ω [57] and nopA::lacZΔp-GmR [17]. All these mutants showed reduced surface motility when compared to HH103 (79.0 ± 3.6 mm). Surprisingly, the rhcJ and rhcV mutants revealed a more severe phenotype (5.0 ± 1.1 and 7.0 ± 2.4 mm, respectively) than those of the ttsI mutants while the nopA mutant exhibited similar surface motility to those of ttsI mutants (13.8 ± 8.7) (Figure 5). A second, independent, mutant in the rhcJ gene (rhcJ::Ω; [58]) behaved as the rhcJ:: Tn5-lacZ mutant (4.9 ± 1.8 mm, Figure 5). Figure 4. Transmission electron microscope (TEM) images of S. fredii HH103 and different mutants. Cells were isolated from the edge of colonies grown on semisolid MM 0.4% agarose in the presence of genistein or ethanol after 24 h of incubation at 28 ◦ C and stained with 2% uranyl acetate. Scale bars correspond to 1 µm. 2.5. A Functional T3SS Is Required for the S. fredii HH103 Genistein-Induced Surface Motility As mentioned above, two independent mutants affected in the ttsI gene exhibited a dramatic reduction in the genistein-induced surface motility of S. fredii HH103, suggesting a role of the HH103 T3SS in this kind of motility. Because of this, we tested the surface motility of HH103 mutants affected in different genes that code for structural components of the S. fredii HH103 T3SS: RhcJ (=NolT), an inner MS ring protein; RhcV (=NolV), a structural protein anchored to the inner membrane; and NopA, the T3SS pilus subunit [ 14 , 17 ]. The mutants analysed were rhcJ::Tn5-lacZ [ 56 ], rhcV:: Ω [ 57 ] and nopA::lacZ ∆ p-Gm R [ 17 ]. All these mutants showed reduced surface motility when compared to HH103 ( 79.0 ±3.6 mm ). Surprisingly, the rhcJ and rhcV mutants revealed a more severe phenotype (5.0 ± 1.1 and 7.0 ± 2.4 mm, respectively) than those of the ttsI mutants while the nopA mutant exhibited similar surface motility to those of ttsI mutants (13.8 ± 8.7) (Figure 5). A second, independent, mutant in the rhcJ gene (rhcJ:: Ω ; [ 58 ]) behaved as the rhcJ:: Tn5-lacZ mutant (4.9 ±1.8 mm, Figure 5). Int. J. Mol. Sci. 2022,23, 7698 9 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 18 Figure 5. Role of different components of the T3SS apparatus on the S. fredii HH103 surface translocation in the presence of genistein. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as gelling agent and scored at 72 h. (A). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. (B). Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). 2.6. Analysing the Putative Role of HH103 Effector Proteins in Genistein-Induced Surface Motility The results mentioned above clearly indicated the essentiality of the HH103 T3SS for genistein-induced surface motility. We wanted to investigate whether the involvement of the HH103 T3SS in genistein-induced surface motility is due to a specific effector delivered by this apparatus or to the apparatus itself. For this purpose, we analysed this kind Figure 5. Role of different components of the T3SS apparatus on the S. fredii HH103 surface translocation in the presence of genistein. Surface motility experiments were performed on minimal medium (MM) using 0.4% agarose as gelling agent and scored at 72 h. ( A ). Box and whisker plots from at least three biological replicates performed with three technical replicates. Asterisks (*) indicate significant differences with the corresponding control sample using the non-parametric test of Mann–Whitney, α = 1%. ( B ). Representative pictures of the motilities exhibited in the presence of genistein by the different S. fredii HH103 mutants analysed. Values under images represent the average and standard deviation of surface migration (given in millimetres and determined as described in the text). 2.6. Analysing the Putative Role of HH103 Effector Proteins in Genistein-Induced Surface Motility The results mentioned above clearly indicated the essentiality of the HH103 T3SS for genistein-induced surface motility. We wanted to investigate whether the involvement of the HH103 T3SS in genistein-induced surface motility is due to a specific effector delivered by this apparatus or to the apparatus itself. 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