Sinorhizobium meliloti GR4 Produces Chromosomal- and pSymA-Encoded Type IVc Pili That Influence the Interaction with Alfalfa Plants.

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. Sinorhizobium meliloti contains two clusters of T4cP-related genes: flp-1 and flp-2, which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with flp-1 are involved in the competitive nodulation of alfalfa plants, but the role of flp-2 remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive S. meliloti GR4 strain and investigated the role of its flp clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double flp-cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA flp clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of flp-1 in the absence of flp-2 reduces the competitiveness for nodule occupation.

Methods for Studying Swimming and Surface Motilities in Rhizobia.

Rhizobia are soil proteobacteria able to establish a nitrogen-fixing interaction with legumes. In this interaction, rhizobia must colonize legume roots, infect them, and become hosted inside new organs formed by the plants and called nodules. Rhizobial motility, not being essential for symbiosis, might affect the degree of success of the interaction with legumes. Because of this, the study of rhizobial motility (either swimming or surface motility) might be of interest for research teams working on rhizobial symbiotic performance. In this chapter, we describe the protocols we use in our laboratories for studying the different types of motilities exhibited by Sinorhizobium fredii and Sinorhizobium meliloti, as well as for analyzing the presence of flagella in these bacteria. All these protocols might be used (or adapted) for studying bacterial motility in rhizobia.

Sinorhizobium meliloti DnaJ Is Required for Surface Motility, Stress Tolerance, and for Efficient Nodulation and Symbiotic Nitrogen Fixation.

Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti.

Analyzing the Effect of Strigolactones on the Motility Behavior of Rhizobia.

In the Rhizobium-legume symbiosis, strigolactones (SLs) promote root nodule formation; however, the exact mechanism underlying this positive effect remains unknown. The recent finding that an SL receptor legume mutant shows a wild-type nodulation phenotype suggests that SLs influence the symbiosis by acting on the bacterial partner. In agreement with this, the application of the synthetic SL analog GR24 on the alfalfa symbiont Sinorhizobium (Ensifer) meliloti has been shown to stimulate swarming, a specialized bacterial surface motility, which could influence infection of legumes by Rhizobia. Surface motility assays for many bacteria, and particularly for Rhizobia, are challenging. The establishment of protocols to study bacterial surface motility is key to decipher the role of SLs as rhizosphere cues for rhizobacteria. In this chapter, we describe a set of protocols implemented to study the different types of motility exhibited by S. meliloti.

2-Tridecanone impacts surface-associated bacterial behaviours and hinders plant-bacteria interactions.

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2-tridecanone (2-TDC), a methylketone (MK) known as a natural insecticide. Application of pure 2-TDC to the wild-type strain phenocopies the free-living and symbiotic behaviours of the fadD mutant. Structural features of the MK determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella-independent motility. Transcriptomic analyses showed that 2-TDC induces differential expression of iron uptake, redox and stress-related genes. Interestingly, this MK also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2-TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2-TDC as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions by interfering with microbial colonization of plant tissues.

Strigolactones in the Rhizobium-legume symbiosis: Stimulatory effect on bacterial surface motility and down-regulation of their levels in nodulated plants.

Strigolactones (SLs) are multifunctional molecules acting as modulators of plant responses under nutrient deficient conditions. One of the roles of SLs is to promote beneficial association with arbuscular mycorrhizal (AM) fungi belowground under such stress conditions, mainly phosphorus shortage. Recently, a role of SLs in the Rhizobium-legume symbiosis has been also described. While SLs' function in AM symbiosis is well established, their role in the Rhizobium-legume interaction is still emerging. Recently, SLs have been suggested to stimulate surface motility of rhizobia, opening the possibility that they could also act as molecular cues. The possible effect of SLs in the motility in the alfalfa symbiont Sinorhizobium meliloti was investigated, showing that the synthetic SL analogue GR24 stimulates swarming motility in S. meliloti in a dose-dependent manner. On the other hand, it is known that SL production is regulated by nutrient deficient conditions and by AM symbiosis. Using the model alfalfa-S. meliloti, the impact of phosphorus and nitrogen deficiency, as well as of nodulation on SL production was also assessed. The results showed that phosphorus starvation promoted SL biosynthesis, which was abolished by nitrogen deficiency. In addition, a negative effect of nodulation on SL levels was detected, suggesting a conserved mechanism of SL regulation upon symbiosis establishment.

ExpR is not required for swarming but promotes sliding in Sinorhizobium meliloti.

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium Sinorhizobium meliloti Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of expR, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that S. meliloti can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional expR gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by S. meliloti is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.