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From 2011.igem.org

Quorum Sensing in Nitrogen-fixing Rhizobia

        In Nitrogen-fixing Rhizobia, we recently found that quorum-sensing play an important role in the symbiosis between the rhizobia and its host plant.
        In the following, we will have a introduction for the three rhizobia we used.

R. leguminosarum bv. viciae




        Early work focused on the rhi system, composed of rhiR (a luxR homolog), rhiI (a luxI homolog), and the rhiABC operon, all of which are located on the symbiotic plasmid pRL1JI. It was demonstrated that rhiABC was controlled by RhiR and that flavonoids repressed the expression of both rhiR and rhiABC. Although the function of rhiABC is unknown, rhiA was shown to be highly expressed in the rhizosphere but not in bacteroids. Moreover, mutations in either rhiA or rhiR led to a decrease in the number of nodules, but only in combination with a nodFE mutant, leading to the hypothesis that the rhi operon may play a role in the early stages of the symbiotic process, as do the nod genes. Further investigation identified rhiI and showed that it was responsible for the synthesis of several short-chain AHLs, including C6-HSL, C8-HSL, and another compound comigrating with C7-HSL.
        In addition to short-chain AHLs, a long-chain AHL, originally termed small for its bacteriocin-like activity, was identified as 3-OH-C14:1-HSL. Initial experiments showed 3-OH-C14:1-HSL to be an inducer of the rhi genes, but later data showing that rhiI and rhiA could be induced by short-chain AHLs (C6-HSL, C8-HSL, and 3-oxo-C8-HSL) but not 3-OH-C14:1-HSL suggested that the induction was indirect. The production of 3-OH-C14:1-HSL is a result of the cinRI locus, located on the chromosome. cinI, the AHL synthase gene, is positively autoregulated by CinR and 3-OH-C14:1-HSL. The mechanism of the regulation of cinR is unclear, since mutations in cinR or cinI or even the lack of the rhi and tra systems do not seem to affect cinR expression. However, cinR expression is cell density dependent. More important, though, is the observation that mutations in cinR and cinI led to decreased levels of all of the short-chain AHLs, suggesting that the cin system is situated at the top of the quorum-sensing network.
        An additional plasmid (pIJ9001)-located locus, raiRI, has recently been identified; this gene synthesizes 3-OH-C8-HSL as its major product, with C6-HSL, C7-HSL, and C8-HSL as minor products. R. leguminosarum RaiI and RaiR have 93 and 88% identity to R. etli CNPAF512 RaiI and RaiR, respectively. As expected, raiI is positively autoregulated by RaiR and 3-OH-C8-HSL but is also induced to a lesser extent by 3-OH-C14:1-HSL and 3-oxo-C8-HSL . These observations help explain the effect of cinRI mutations on the production of some of the short-chain AHLs and also suggest that an additional 3-oxo-C8-HSL-producing locus may contribute to regulation of raiRI.
        Recent work has also identified a cluster of genes on pRL1JI with homology to the trb operon of A. tumefaciens. Wilkinson et al. identified traI (an AHL synthase), followed by the trb genes (which function in mating pore formation), as in A. tumefaciens and Rhizobium NGR234. Interestingly, in addition to a traR regulator, R. leguminosarum carries a second regulatory gene, bisR, downstream of the trb operon. BisR has 59% identity to CinR, while TraR has 64% identity to NGR234 TraR. Furthermore, traR expression seems to be controlled by cinRI through the BisR regulator, presumably through the binding of BisR to 3-OHC14:1-HSL. TraR then goes on to control the conjugal transfer of pRL1JI by inducing the trb operon. Unpublished data from the laboratory of Alan Downie also suggests that traI is responsible for the synthesis of 3-oxo-C8-HSL, as in A. tumefaciens and NGR234, which could link activation of the tra system to a small induction of the rai system. Another link in the regulatory network is the observation that BisR can repress cinI, suggesting a negative-feedback loop between the cin and tra systems.
        Although much work has gone into characterizing the quorum-sensing network of R. leguminosarum, little is known about the role of these systems in the life cycle of the organism. Mutations in the rai, cin, and tra systems do not have any apparent defects in nodulation. The rhi system seems to play a role in nodulation efficiency, but no dramatic defect has been observed for rhi mutants that might suggest a possible mechanism. The only system with a defined role is the tra system, since it was clearly shown to regulate the conjugal transfer of pRL1JI, a symbiotic plasmid. However, the advantage of having plasmid transfer under the control of the cin system is not apparent. Lastly, the growthinhibitory role of 3-OH-C14:1-HSL and cinRI is still a mystery. This AHL-mediated growth inhibition was shown by Gray et al. To result from an early induction of the stationary phase, but only strains carrying pRL1JI are sensitive to the growth inhibition. Furthermore, addition of OH-C14:1-HSL has been shown to promote starvation survival of R. leguminosarum cultures that enter stationary phase at low cell density.




R. etli CFN42
        R. etli strain CFN42 contains one chromosome and six plasmids (p42a to p42f). Plasmid p42a is self-transmissible at a high frequency (10-2) and is required for mobilization of the symbiotic plasmid, p42d. Recently, a traI-trb operon, with high similarity to the transfer genes of pTi and pNGR234a, has been localized on p42a. Four regulatory genes, traI, traR, cinR, and traM, have also been found on this plasmid (Fig. 5).



        Only two AHLs have been described in R. etli CFN42, 3-oxo-C8-HSL, synthesized by TraI, and a putative 3-OH-C8-HSL, whose function and synthase remain to be identified.
        Transfer of p42a is regulated by the products of traI, traR, and cinR. TraI expression is dependent on itself and on the presence of TraR and CinR. The traR gene seems to be expressed constitutively, but expression of cinR requires an active TraI. The p42a plasmid also encodes a putative TraM-like antiactivator, but no expression of this gene was detected under the experimental conditions tested. Therefore, it seems that conjugal transfer of p42a is derepressed, which could account for its high transfer frequency. The R. etli CFN42 quorum-sensing system does not seem to be directly involved in the symbiotic process (derivatives of CFN42 lacking p42a are able to effectively nodulate bean plants) , but it probably plays an indirect role by regulating the conjugative transfer of the p42d symbiotic plasmid.
S. meliloti Rm1021
        The well-characterized S. meliloti strain Rm1021 harbors at least two quorum-sensing systems (Fig. 7) . The sinR/sinI locus is responsible for the production of several novel AHLs, ranging in size from C12-HSL to C18-HSL. Some of these are the longest AHLs identified so far. Disruption of the sin system correlates with a delay in the appearance of nitrogen-fixing nodules, as well as with an overall decrease in the number of pink nodules, suggesting a role for quorum sensing in establishing a successful symbiosis with Medicago sativa. More recently, it was shown that the sinR and sinI genes were required for synthesis of EPS II by a strain proficient in the production of this exopolysaccharide. The Rm1021 strain, which normally does not produce EPS II, has an insertion sequence that results in the disruption of expR (a luxR homologue). Strains proficient in EPS II production, such as Rm41 and Rm8530 (Rm1021 expR_), possess an intact expR gene. ExpR is a positive regulator of the exp genes, which are responsible for EPS II biosynthesis; however, it is unclear whether this regulatory effect is direct or indirect. In a sinI mutant, expression of several of the exp genes is abolished, and this deficiency can be fully complemented by the addition of either crude AHL extracts from wild-type Rm1021 or synthetic C16:1-HSL. Therefore, it seems that the sinRI locus controls EPS II production via ExpR. Regulation of EPS II production by sinRI was shown to be important for nodule invasion, since a strain that produces exclusively EPS II, combined with a sinI mutation, is no longer capable of forming nitrogen-fixing nodules. These results provide the first details of a mechanism for quorum sensing controlin the development of symbiosis.