Elsevier

Plasmid

Volume 106, November 2019, 102443
Plasmid

Plasmid pSfr64a and the symbiotic plasmid pSfr64b of Sinorhizobium fredii GR64 control each other's conjugative transfer through quorum-sensing elements

https://doi.org/10.1016/j.plasmid.2019.102443Get rights and content

Highlights

  • Transfer of pSfr64a is regulated by quorum-sensing elements encoded in pSfr64a, pSfr64b and the chromosome.

  • Transfer of pSfr64b is regulated by quorum-sensing elements encoded in pSfr64a, pSfr64b and the chromosome.

  • The oriT from pSfr64a from Sinorhizobium fredii GR64 can be processed by the relaxase from pRet42a of Rhizobium etli CFN2.

  • The oriT from pRet42a from Rhizobium etli CFN42 can be processed by the relaxase from pSfr64a of Sinorhizobium fredii GR64.

Abstract

Rhizobia are nitrogen-fixing symbionts of plants. Their genomes frequently contain large plasmids, some of which are able to perform conjugative transfer. Plasmid pSfr64a from Sinorhizobium fredii GR64 is a conjugative plasmid, whose transfer is regulated by quorum sensing genes encoded by itself (traR64a, traI64a), in the symbiotic plasmid pSfr64b (traR64b, traI64b), and in the chromosome (ngrI). Also, transfer of pSfr64b requires quorum sensing elements encoded in this plasmid (traR64b, traI64b), in pSfr64a (traR64a), and in the chromosome (ngrI). These results demonstrate that pSfr64a and the symbiotic plasmid depend on each other for conjugative transfer. Plasmid pSfr64a from S. fredii GR64 is unable to transfer from the genomic background of Rhizobium etli CFN42. Our results show that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa, underlining their functional similarity and suggesting that in addition to the external signals, the “cytoplasmic environment” may pose a barrier to plasmid dissemination, even if the plasmids are functional in other aspects.

Introduction

The participation of conjugative transfer in events that lead to evolution and diversification has been highlighted (Wiedenbeck and Cohan, 2011). Among soil inhabitants, bacteria belonging to the Rhizobiaceae have some attractive features, such as the capacity to establish a symbiotic relationship with the roots of legume plants and the presence of multipartite genomes, usually formed by a chromosome and a variable number of large plasmids (López-Guerrero et al., 2012). Although there is phylogenetic evidence of horizontal transfer of genomic information (Sullivan and Ronson, 1988; Pappas and Cevallos, 2011), not much is known about the conjugative transfer of rhizobial plasmids.

Rhizobium etli establishes symbiosis with the roots of bean (Phaseolus vulgaris) plants. The R. etli type strain, CFN42, isolated in México, contains one chromosome and six large plasmids, pRet42a to pRet42f, ranging in size from 194 to 642 Kb. Plasmid pRet42d corresponds to the symbiotic plasmid (pSym), because it carries the genes required for nodulation and nitrogen fixation necessary for a successful symbiosis (González et al., 2006).

Analysis of the conjugative transfer ability of the plasmids of R. etli CFN42 showed that pRet42a is a conjugative plasmid, regulated by quorum sensing, similar to the Ti plasmid of Agrobacterium tumefaciens (Piper et al., 1993). pRet42a contains a transfer region adjacent to the replication genes. This region contains the genes required for DNA processing, transfer and replication (Dtr), the genes involved in the mating pair formation (Mpf), and genes involved in regulation: traI, traR and cinR. The quorum sensing regulation is mediated by the product of a traI gene, encoding an acyl homoserine lactone synthase, and two transcriptional regulators: traR and cinR (Table 1). When cellular density is high, the acyl homoserine lactone (AHL) inside the cells forms complexes with the TraR and CinR regulators, which are able to induce transcription of the Dtr and Mpf genes involved in conjugation (Tun-Garrido et al., 2003).

The symbiotic plasmid pRet42d from R. etli CFN42 can be mobilized through conduction, by cointegrating with pRet42a. This cointegration can be achieved by RecA-mediated homologous recombination, or through IntA-mediated site-specific recombination (Brom et al., 2004; Hernández-Tamayo et al., 2013). The pSym is also able to achieve mobilization independent of pRet42a, employing transfer genes located in pRet42d, which are only expressed when the RctA repressor is inactivated by mutation (Pérez-Mendoza et al., 2005; Nogales et al., 2013).

We have started to analyze other bean-nodulating strains. Sinorhizobium fredii strain GR64 was isolated from bean nodules in Granada, Spain (Herrera-Cervera et al., 1999) (Table 1). This strain contains a transmissible plasmid (pSfr64a). The sequence of this plasmid contains 166 ORFs: 38 are similar to sequences from the chromosome of S. fredii strain NGR234, 51 are similar to genes from pRet42d and 36 are similar to sequences from pRet42a of R. etli. Plasmid pSfr64a was found to be required for transfer of the pSym (pSfr64b) of the S. fredii strain (Cervantes et al., 2011). Although the transfer genes of pRet42a from R. etli and pSfr64a from S. fredii are highly similar (López-Fuentes et al., 2015, Fig. 3A), pSfr64a is unable to transfer from the R. etli genomic background.

In the literature, two main types of conjugative transfer regulation systems have been described for rhizobial plasmids. One is a cell-density dependent quorum sensing mechanism, and the other is based on RctA-mediated repression (Ding and Hynes, 2009; Nogales et al., 2013). In addition to R. etli CFN42, quorum sensing regulated transfer has been described in Rhizobium leguminosarum bv viciae (Danino et al., 2003), and in the symbiotic island of Mesorhizobium loti R7A (Ramsay et al., 2009). All of them depend on luxI-luxR type regulators, although there are variations particular to each plasmid (McAnulla et al., 2007; Khan and Farrand, 2009). Recently, the participation of other elements affecting the transfer ability of diverse plasmids has been described (Ding et al., 2013; Pistorio et al., 2013; Ramsay et al., 2013).

In order to further understand the mechanisms leading to plasmid distribution and diversification in bean-nodulating rhizobia, in this work we analyzed the regulatory mechanism governing conjugative transfer of plasmids pSfr64a and the pSym pSfr64b from S. fredii strain GR64.

Section snippets

Bacterial strains and plasmids

A summary of the relevant features of R. etli CFN42 and S. fredii GR64 are presented in Table 1. The bacterial strains and plasmids used in this work are described in Table 2. Rhizobium strains were grown at 30 °C on PY medium (Noel et al., 1984). Escherichia coli and Agrobacterium tumefaciens strains were grown on Luria-Bertani medium (Miller, 1972) at 37 and 30 °C, respectively. When required, antibiotics were added at the following concentrations (μg/ml): nalidixic acid, 20; spectinomycin,

Quorum sensing elements encoded in plasmids pSfr64a, pSfr64b and in the chromosome regulate transfer of pSfr64a from S. fredii

The presence of traI and traR genes on pSfr64a suggests that the conjugative transfer of this plasmid may be regulated by quorum sensing elements. We have completed a draft genome sequence of S. fredii GR64 (Torres Tejerizo et al., 2012). The sequence shows that the symbiotic plasmid pSfr64b contains a complete set of transfer genes. The similarity of these genes to those located on pSfr64a is shown in Fig. 1. Also, the chromosome showed the presence of two genes related to quorum sensing.

Discussion

Here we show that conjugative transfer of plasmids pSfr64a and pSfr64b from S. fredii is regulated by quorum sensing elements, similar to other rhizobial plasmids (Tun-Garrido et al., 2003; Danino et al., 2003). In this case, transfer is modulated by regulators encoded in three different replicons: pSfr64a, the symbiotic plasmid pSfr64b, and the chromosome. The participation of regulatory elements located on different replicons has been shown in other instances. For example, in A. tumefaciens,

Author contributions

LC carried out most of the molecular genetics experiments. GTT and FM-S participated in the design and performed some of the molecular genetics. DR participated in the design of the study and in the discussion of results. SB conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

Funding

This work was partially supported by grant 188967 for Bilateral Mexico-Argentina Cooperation, from CONACYT-CONICET, by grant IN203515, from PAPIIT, DGAPA, UNAM, and by grant PICT2016-0210 to G.T.T. G.T.T. is member of the Research Career of CONICET and has received a fellowship from the Alexander von Humboldt Foundation.

Declaration of Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgements

We are grateful to Paul Gaytán and Eugenio López for synthesis of oligonucleotides.

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