https://orcid.org/0000-0002-6773-0732
Correction
24 Jan 2025: The PLOS One Staff (2025) Correction: Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules. PLOS ONE 20(1): e0318411. https://doi.org/10.1371/journal.pone.0318411 View correction
Figures
Abstract
Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.
Citation: Luchetti A, Castellani LG, Toscani AM, Lagares A, Del Papa MF, Torres Tejerizo G, et al. (2023) Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules. PLoS ONE 18(5): e0285505. https://doi.org/10.1371/journal.pone.0285505
Editor: Günther Koraimann, University of Graz, AUSTRIA
Received: December 8, 2022; Accepted: April 25, 2023; Published: May 18, 2023
Copyright: © 2023 Luchetti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The minimal data set underlying the results described in our paper can be found under Genbank accession number MZ505104 (https://www.ncbi.nlm.nih.gov/nuccore/MZ505104).
Funding: G.T.T. - PICT2020-02314 - Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación - https://www.argentina.gob.ar/ciencia/ agencia M.P. - PICT2017-2833 - Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación - https://www.argentina.gob.ar/ciencia/agencia G.T.T - PIP0678 - Consejo Nacional de Investigaciones Científicas y Técnicas - https://www.conicet.gov.ar/ We acknowledge support for the publication costs by the Open Access Publication Fund of Bielefeld University and the Deutsche Forschungsgemeinschaft (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bacteria are ubiquitous microorganisms able to colonize and adapt to different environments in a very short-term period. Horizontal gene transfer (HGT) plays a key role in the adaptation process, being conjugative plasmid transfer one of the most efficient DNA exchange mechanisms between bacteria [1]. Plasmids are widely distributed selfish genetic elements that are characterized by their autonomous replication. It has been shown that plasmids have many strategies to remain stable within host cell populations [2–4]. Plasmids consist of backbone genes that are involved in replication, stability and in some cases conjugative transfer, in addition to accessory genes that confer variable traits that might give an advantage to their hosts, such as antibiotic resistance, genes involved in the degradation of diverse carbon sources or in the establishment of pathogenesis or symbiosis [5].
Gram-negative bacteria belonging to the genera Rhizobium, Sinorhizobium, and Mesorhizobium, among others, can grow in the soil in free-living conditions and in symbiosis with the root of leguminous plants as nitrogen-fixing organisms. A common feature of the genomes of these genera is that, in addition to the chromosome, they usually harbor plasmids encoding widely diverse functions [6–8]. Rhizobial plasmids vary greatly in number (1 to 10) and size (ten to thousands of kilobases) and can constitute a high proportion of the bacterial genome [6, 7, 9]. Many of them have relevant roles during the interaction between rhizobia and the host plant. Most of the genes required for the symbiotic process are encoded in the so-called symbiotic plasmids or pSym [10]. In addition to these pSym, most of the plasmids carried by rhizobia are dispensable for symbiosis or have simply not yet been assigned a specific function. These plasmids with no apparent function are referred, in a generic manner, as non-symbiotic, cryptic, or accessory plasmids [7].
For the survival of plasmids in the host cell, they must replicate through a functional replication system, independent of the chromosomal replication system. In addition, different mechanisms involved in plasmid stability have been described in the literature, such as multimer resolution, active partitioning and post-segregational killing, also known as toxin-antitoxin or plasmid addiction systems [2–4]. Regarding replication systems, necessary to achieve independent replication, the best-studied system in soil bacteria is the repABC replication system [11]. In the Rhizobiaceae family, specifically, repABC-type replicons predominate in plasmids in which their replication regions have been studied [12, 13]. The repABC system consists of four genes: an operon composed of three genes (repA, repB, and repC) [14–18] and a gene encoding a small regulatory antisense RNA (ctRNA) located in the complementary strand of the intergenic space between repB and repC [19–22]. RepA and RepB belong to the ParAB family of partition proteins. The interaction between RepA, RepB and a centromere-like sequence (parS) provides the plasmid’s segregation machinery [11, 13, 23]. The parS site of repABC plasmids consists of single or multiple 16 bp palindromic sequence that could be located at different positions in each plasmid [24–26]. Regarding RepC, it has been proposed to be the initiator protein [15]. Cervantes-Rivera et al. [27] showed that in the repABC operon of pRetCFN42d of Rhizobium etli CFN42, repC is the only required element to sustain replication. In addition, the origin of replication of the repABC plasmids is located at the central section of the repC gene [27, 28]. Incompatibility determinants are also commonly present in plasmids. For repABC plasmids, three main incompatibility determinants were described [20, 24, 25, 29–31]. In addition to being involved in plasmid segregation, the 16 bp palindromic sequence parS exerts strong incompatibility towards the parental plasmid or plasmids having identical parS [24, 25]. The other elements involved in plasmid segregation, RepA and RepB, could induce plasmid incompatibility when expressed in trans, downregulating the transcription of the repABC operon [29, 31]. Another incompatibility determinant consists of the ctRNA located between repB and repC. It has been demonstrated that the expression of this small RNA in trans causes the loss of the parental plasmid due to the interference with the expression of repC [20, 25]. Recently, Rivera-Urbalejo et al. [30] mapped the sequences of the ctRNA that are required for plasmid incompatibility by site directed mutagenesis. Besides the repABC system, other replication systems have been described in rhizobia. The second system of replicons, called the repC family, is evolutionarily related to the repABC family. These systems share the replication initiation protein RepC, but in contrast to the organization of repABC, repC is not forming an operon with repA and repB genes [32]. In this family, an antisense RNA also plays a central role as a negative regulator of repC expression and as a determinant of incompatibility [33]. A third system, includes only one member, the 7.2 kb pRm1132f plasmid isolated from S. meliloti strain 1132, which also belongs to group II of rolling circle replication systems [34].
As we mentioned before, the segregational stability of the rhizobial plasmids is achieved by the activity of plasmid-specific partitioning proteins (RepAB) that direct plasmid copies to new daughter cells during cell division. In addition to partitioning systems, plasmids can harbor toxin-antitoxin (TA) genes to ensure the inheritance of plasmids. TA systems are small genetic modules coding for a stable toxin and for an unstable antitoxin protein that counteracts the activity of the toxic protein. Wheatley et al [35], described the presence of a TA module on Rhizobium leguminosarum bv. viciae 3841 pRL10 plasmid and associate its presence to the inability to lose pRL10.
In our laboratory, we have been studying rhizobial accessory plasmids as vehicles of adaptation and evolution [36–41]. We have previously described the transmissibility properties of two cryptic plasmids from the strain Sinorhizobium meliloti LPU88, a local isolate from Argentina. One of them, pSmeLPU88b, resulted to be mobilizable only if helper functions were supplied in trans by the accompanying plasmid pSmeLPU88a (binary conjugal system) [41]. Later, it was shown that the mobilization region of plasmid pSmeLPU88b presented a new mob region that was conserved in other rhizobia and in different Gram-negative bacteria [42]. To get a deeper insight into the traits encoded by this plasmid we report here the complete sequence and the genomic characterization of pSmeLPU88b. Moreover, we studied the replication and stability elements found in it. Plasmid pSmeLPU88b presents two origins of replication, one belonging to the repABC family and another related to the repC family. Interestingly, both are capable of supporting the replication of the plasmid. It is noteworthy that the pSmeLPU88b plasmid backbone is not only conserved in S. meliloti plasmids but also in Sinorhizobium spp. and Rhizobium spp. strains thus, our work depicts replication genes distributed in rhizobia and contributes to the basic knowledge of rhizobial plasmid biology.
Materials and methods
Bacterial strains and plasmids
The strains and plasmids used are listed in Table 1. Escherichia coli was grown on LB [43] medium at 37°C. S. meliloti strains were grown on TY [44] medium at 28°C. For the agarized media 15 g of agar per liter of medium was added. The growth kinetics (28°C, 200 rpm) was analyzed by monitoring OD600 in a microplate reader (BMG Labtech, Germany). The final antibiotic concentrations per mL of medium were: 25 μg kanamycin (Km) and 10 μg gentamycin (Gm) for E. coli; 400 μg streptomycin (Sm), 120 μg neomycin (Nm), and 30 μg Gm for S. meliloti.
DNA sequencing and bioinformatics tools
The pSmeLPU88b plasmid sequence was obtained by sequencing S. meliloti LPU88 at SNPsaurus (https://www.snpsaurus.com/illumina-bacterial-assembly/) by means of Illumina technology. The reads obtained were assembled using Spades Assembler software (Version 3.12.0). Plasmid finishing was accomplished using a previously sequenced plasmid library [42]. The accession number for plasmid pSmeLPU88b is MZ505104. Plasmid pSmeLPU88b was automatically annotated by the genomes annotations platforms Prokka [48] and RAST [49] and then manually curated. Sequence alignment and graphic comparison were performed using the genome comparison visualizer Easyfig [50]. For the identification of ctRNAs present in pSmeLPU88b plasmids, the intergenic region between the repB and repC genes of repABC family and the region upstream of repC of repC family were aligned along with the ctRNA genes from some repABC and repC families. The transcriptional start sites of p42d [20] and pRmeGR4a [33] have been previously experimentally determined. We used that information to map the ctRNAs. The ctRNA structures were predicted using the RNAfold WebServer (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi).
Bacterial mating
Bacterial matings were performed as described by Simon et al [45]. Stated in brief, liquid cultures were grown to the early exponential phase for donor cells and the late exponential phase for recipient cells. Donor and recipient strains were mixed in a 1:1 volume ratio and plated on TY plates, and then incubated overnight at 28°C. Afterwards, the transconjugants and controls were plated in TY medium supplemented with the appropriated antibiotics.
Plasmid profiles—Eckhardt gels
Cells were grown in TY medium to the mid-exponential phase and the plasmid profiles were performed according to previously established techniques [51, 52].
DNA manipulation and genetic constructs
Plasmid and total DNA preparation, restriction-enzyme analysis, cloning procedures, and E. coli transformation were performed as previously described [53].
Construction of repC mutants
An internal fragment of each repC gene was amplified with the appropriate primers (S1 Table) with Pfu polymerase (for repC1: repC1-(ABC)-MN, repC1-(ABC)-MC; for repC2: repC2-MN repC2-MC) (S1 Table). The obtained fragment was cloned in the SmaI site of pK18mob (a suicide vector in rhizobia) resulting in the vectors pKrepCmut and pKrepABCmut. The obtained plasmids were transferred by conjugation to strain LPU88 to yield the site-specific insertional mutagenesis. The insertion was evaluated by PCR with external primers: repC1-(ABC)-Ext and repC2-trunc for repC1 and repC2, respectively (S1 Table).
Functional analysis of replication modules
RepC family replicon. The complete coding sequence of repC gene and 500 bp upstream was obtained using primers repC-F and repC-R (S1 Table) amplifying a 2,492 bp fragment with Pfu polymerase. This fragment was cloned in the SmaI site of pK18mob (this vector is not able to replicate in rhizobia). The constructed plasmid, designed pKrepC, was introduced into S. meliloti strains by conjugation, using E. coli S17-1 as the donor, to determine if this plasmid was able to replicate in rhizobia and if it is incompatible with different plasmids.
RepABC family replicon. We have previously described the obtention of a partial library of pSmeLPU88b plasmid [42, 54]. One clone of the library, pG700, contained a DNA fragment encoding the C-terminal domain of the RepC of the repABC operon in pG18mob2 plasmid (non-replicative in rhizobia). pG700 was introduced into strain LPU88 by conjugation. The presence of sequences in common between pG700 and pSmeLPU88b enabled the formation of cointegrate plasmids. The entire repABC operon was then obtained by isolation of total DNA, digestion of isolated genomic DNA with restriction endonuclease SmaI, to generate a restriction fragment containing the integrated plasmid and repABC operon. Circularization of this DNA fragment by ligation and transformation into E. coli DH5α competent cells allow us to obtained plasmid pGrepABC.
Truncated RepC2. By introducing a stop codon in the reverse primer (repC2-trunc, S1 Table), a truncated variant of the repC2 gene was amplified by PCR and cloned in the SmaI site of the pK18mob suicide plasmid. The position of the reverse primer was selected after aligning the DNA sequence of repC2 of LPU88 to other repC family sequences.
parS site. In the non-replicative pG700 plasmid the parS site was identified. To test its function, pG700 plasmid was digested with EcoRI and the liberated fragment cloned in the replicative plasmid pBBR1MCS-2, yielding pBBRparS.
Stability of plasmids
The stability of plasmids in S. meliloti LPU88 and S. meliloti 2011 was determined as previously described [55]. The proportion of bacteria that harbor plasmids was determined by plating subculture steps on TY agar and further replication on TY agar plates and TY agar plates containing the corresponding antibiotics for each plasmid. Colonies that had lost the plasmid were able to grow on TY agar plates but not in TY agar plates with antibiotics, while colonies harboring plasmids were able to grow on both plates. The rate of plasmid loss was calculated.
Results and discussion
Overall features of pSmeLPU88b
We have previously reported the conjugal properties of two cryptic plasmids from the strain S. meliloti LPU88 [41]. To further advance in the characterization of the mobilizable plasmid pSmeLPU88b, the complete nucleotide sequence was obtained and analyzed, focusing on gene content and backbone gene organization. General features of pSmeLPU88b sequence revealed the following: the plasmid genome is 35,933 bp long and its GC content is 58.6%. The annotation of the plasmid nucleotide sequence was first done automatically by Rast [49] and Prokka [48] followed by manual curation, resulting in 31 CDS (Fig 1A, Table 2). The 31 CDS presented identity to genes in the NCBI nr database and 25 of them (78%) could be assigned to a Cluster of Orthologous Group (COG) (Table 2). Two of the remaining six CDS could be assigned to a PFAM family while four CDS could not be assigned to any specific function.
A. Schematic plot of pSmeLPU88b. From the inner to the outer circle: genomic position in kb; GC skew; GC content; predicted protein-coding sequences (CDS). The repA gene was chosen as the first gene. The plot was made using https://proksee.ca/. B. Structural comparisons of pSmeLPU88b and pMBA9a replication regions. The arrows indicate genes and the blue rectangle ISRm17.
Of the total CDS, eleven were associated with plasmid biology functions, like replication (repABC1 and repC2), stability (CDS 4,5,6), and mobilization (parA-like, mobCZ, traI). Previously, Giusti et al, [42] characterized the CDS parA-like and mobCZ, and these were found to be associated with the DNA-transfer-and-replication region (Dtr) of pSmeLPU88b (Table 2). The other identified CDS encode proteins associated with different metabolic processes (CDS18, CDS19, CDS25, CDS26, CDS27) like amino acid metabolism (CDS14, CDS17) or oxidoreduction (CDS 13, CDS15, CDS19, CDS20), transcriptional regulation (CDS16, CDS23) and transposition (CDS28, TRm17) (Table 2). Five CDS encoded for hypothetical proteins (CDS4, CDS8, CDS9, CDS21, CDS22) that are also found in other S. meliloti strains, but for which no other inferences could be made (Table 2).
In silico analysis of the replication and stability modules
The in silico analysis of the obtained sequence data allowed us to identify two plasmid replication modules: one belonging to the repABC family [13], and the other related to repC family [32]. The DNA alignment of both regions resulted in nearly identical elements to the ones present in the plasmid pMBA9a from an S. meliloti isolate from Canada (99% DNA identity) [56]. However, while the repC and repABC modules present in pMBA9a are contiguous, in the plasmid pSmeLPU88b are separated by the IS-element ISRm17, which belongs to the group ISDoll of the ISCNY family (Fig 1B). The presence of more than one type of replication system in the S. meliloti cryptic plasmids seems to be a common feature since it has also been observed in other plasmids like pRmeGR4a, pRmeGr4b, pSmeSM11a, pHRC017, and the paccessoryA of strains HM006, T073 and USDA 1157. This feature is not only limited to S. meliloti species since pSF45436e from Sinorhizobium fredii CCBAU45436, plasmid A from Sinorhizobium americanum CCGM7, pRtrCIAT899b from Rhizobium tropici CIAT899 and pAtS4a from Agrobacterium vitis S4 present two types of replicons. In addition, the presence of two replicons seems to be a common feature in alphaproteobacteria. Bartosik et al. [18] described a plasmid of Paracoccus versutus UW1 containing repC and repABC systems. Later, the same authors found several strains of Paracoccus pantotrophus containing four plasmids with both types of replication machinery [16].
Usually, RepC proteins from repC family (PRK13824 domain) present a size of ca. 400 amino acids long (https://www.ncbi.nlm.nih.gov/Structure/sparcle/archview.html?archid=11486891), but it is noteworthy that RepC2 from pSmeLPU88b has 611 amino acids. There are only three entrances in the NCBI database with a RepC protein with the same characteristics, all of them of S. meliloti strains. Specifically, in pMB9a of strain MB9A, and in DNA contigs from strains USDA 1027 and USDA 1508, all of them sharing 99% amino acid identity.
A general characteristic of repABC operons is the presence of a conserved intergenic region between the repB and repC genes. In this region, a gene coding a 55–59 nt small non-translated RNA (ctRNA) in the opposite orientation to the repABC operon is usually located, which could generate incompatibility with its parental plasmid if it is introduced in trans [19, 20]. A similar antisense RNA was described for the repC family [33]. These ctRNA not only play a role as an incompatibility factor but also as a negative regulator of repC gene expression [33]. We identified in silico ctRNA, upstream of each repC gene. The computer-predicted secondary structure of both ctRNA presented similar structures as those previously described (S1 Fig) [19, 20, 22, 33]. An alignment of ctRNAs of other rhizobial plasmids showed that ctRNArepABC presents a higher sequence conservation than ctRNArepC, and the last one is identical to the ctRNA present in pMB9a and the contigs from USDA 1027 and USDA 1508 strains (S2 Fig).
The centromere-like DNA sequence parS consist of one or more copies of a 16-bp palindromic consensus sequence GGTNNGNGCNCNNACC close to the repABC genes [13]. The parS locus could be located downstream from repC [15], within the repA-repB intergenic zone [57], or upstream from repA [25]. Recently, Czarnecki et al. [26] showed that in the repABC system of plasmid pAMI4 of Paracoccus aminophilus, in addition of a single parS near the repABC genes, the plasmid contains three additional parS repeats, 11.5 kb downstream of repC. We searched for the presence of parS in pSmeLPU88b and found a 16-bp palindrome, 93 bp downstream of repC1 gene. The palindrome sequence CCTGTCAGCTGACAGG is partially conserved compared to the consensus sequence.
Downstream of the repABC replication system are located three CDS homologous to recombinases and toxin-antitoxin (TA) systems. The recombinase (CDS 5, XerD) presents its maximum amino acid identity (96%) with the protein CDO22_34430 (ASQ15018) of plasmid accessory A of S. meliloti HM006. The CDS 5 belongs to the Cre-like recombinases, a tyrosine-based site-specific recombinase. These enzymes are associated with plasmid maintenance by mediating the recombination site between the repeated sequences of the concatemer. Plasmid dimers could be formed through homologous recombination and, since a dimer has two origins of replication, the copy number control system perceives two entities, but only one plasmid is available for segregation [58]. In Rhizobium etli CFN42 it was described a recombinase RinQ, which is required to exert incompatibility to pRetCFN42d plasmid. RinQ is also near to the repABC operon but belongs to the invertase/resolvase family (γδ family) [59]. In pLPU88b, the TA system is formed by CDS 6 and CDS 7 (designated papA and papT respectively for plasmid addiction protein). PapA shown 100% amino acid identity with the CDO22_34435 (accessory A plasmid of S. meliloti HM006) and CN144_34000 (contig 185 of strain USDA 1508). PapT presented 100% amino acid identity to CN144_33995 (contig 185 of strain USDA 1508). PapA belongs to the type II toxin-antitoxin systems (pfam02604, Antitoxin Phd_YefM), members of this family bound to their toxin partners, and they can bind DNA repressing the expression of TA operons. PapT possesses a conserved putative domain belonging to the PemK superfamily, which inhibits the growth of host cells. PemK is responsible for mediating cell death by inhibiting protein synthesis through the cleavage of single-stranded RNA. This system is completed by the antitoxin PemI that inhibits the action of the PemK toxin [60]. PemI belongs to the MazE_antitoxin family (pfam04014), a family different from the PapA antitoxin family.
It is noteworthy that the repABC and the three CDS observed downstream presented the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Apparently, this genetic organization is a common feature in S. meliloti, S. medicae, and there are representatives in S. fredii, S. americanum, S. arboris and Rhizobium favelukesii. In addition, in some plasmids, there is also present a repC family replicon (Fig 2). This region could be the so-called “backbone” of a plasmid, which generally encodes functions involved in replication, maintenance, and in conjugative plasmid transfer. As we mentioned before, plasmids containing repC and repABC systems could be found in other alphaproteobacteria. The search for complete plasmid sequences in databases showed that plasmids pAK2 of Paracoccus sp. AK26, pBM151 of P. tegillarcae BM15, pZO2 of P. zhejiangensis J6, plasmid a of Rhodobacter sphaeroides MBTLJ-20 and plasmid D of R. sphaeroides 2.4.1, among others, present both types of replication systems. However, in these plasmids the recombinase and TA systems are not found in the neighborhood of the repABC system. Furthermore, the two origins of replication were located further apart from each other compared to the distance in rhizobial plasmids.
Genetic organization of pSmeLPU88b replication region compared with regions present in other rhizobial plasmids. Relevant orthologs are marked with the same color.
Functional characterization of the two-replication modules of plasmid pSmeLPU88b
As we mentioned before, rhizobial strains contain several plasmids and each one of them carries genes of the repABC or repC families. The most obvious case of this peculiarity is observed in R. etli CFN42 and R. leguminosarum 3841, each of which has six plasmids all belonging to the repABC family. This suggests that different repABC plasmids belong to different incompatibility groups [61, 62]. Furthermore, replicons harboring two repABC operons have also been found [61, 62]. In other examples, plasmids with one repC and one repABC were observed [56, 63, 64]. This condition has been proposed as beneficial [65], assuming that the presence of more than one replication module can contribute to the overall stability of the plasmid that carries them.
To determine the functionality of the repABC and repC replicons present in pSmeLPU88b, we carried out two strategies: on the one hand, we made site-specific insertional mutagenesis on each of the replication initiation proteins; in a second approach, each of the replication modules was cloned separately in vectors that originally are suicide in S. meliloti, but with the replication module became replicative. In both assays, the ability to maintain replication in rhizobia was evaluated. We could obtain mutants in both repC genes by insertional mutagenesis, demonstrating the functionality of both replicons. After cloning each replication module in the suicide vectors, the resulting pKrepC and pGrepABC (Table 1) plasmids were transferred to S. meliloti 2011 and S. meliloti LPU88 strains. To verify the presence of plasmids pKrepC and pGrepABC in rhizobia, transconjugants were taken randomly and analyzed by Eckhardt-type gels. The replication functionality of the new build replicons has been first evaluated through the presence of the expected plasmid in S. meliloti 2011 (S3 Fig) and in S. meliloti LPU88 (S4 Fig).
Another interesting aspect to mention is the incompatibility of the vectors pKrepC and pGrepABC with respect to the plasmid pSmeLPU88b. Incompatibility between plasmids is the inability of two plasmids to coexist within the same cell. In general, this situation is the result of sharing certain elements involved in plasmid replication or segregation, as well as in some regulatory components of these functions. Strikingly, when plasmids pKrepC and pGrepABC were transferred independently by conjugation to LPU88 strain, pSmeLPU88b was removed from the cells (S4 Fig). The analysis by Eckhardt-type gels confirmed that both replicons could exclude plasmid pSmeLPU88b (S4 Fig). It should be noted that selection pressure with antibiotics -Nm (for pKrepC) and Gm (for pGrepABC)-, forces the maintenance of the introduced plasmids instead of the resident plasmid pSmeLPU88b. This result was surprising because it was expected that pSmeLPU88b would replicate by the different replication system as the one introduced. The observed behavior shows the existence of certain interactions between both replication systems. To corroborate this hypothesis, we transferred the plasmid pGrepABC to the S. meliloti 2011 derivative previously containing pKrepC, and the plasmid pKrepC to the S. meliloti 2011 derivative previously containing pGrepABC (S3 Fig). In the S. meliloti 2011 background, the interaction/interference between the replicons was not observed since they could replicate independently in most of the cases, but in a few cases a cointegration between pKrepC and pGrepABC was observed (S3 Fig).
As we saw that each replication module was functional, the maintenance of the plasmids was tested. First, the repC mutants were evaluated. S. meliloti LPU88 cells carrying pSmeLPU88::pKmutC or pSmeLPU88::pKmutABC (mutants in each module) were grown in serial cultures in a nonselective medium, and plasmid loss rates were measured by plating to determine the proportion of cells retaining the plasmid (see Materials and Methods). Both plasmids were stably maintained in progeny cells since 100% of the cells retained the plasmid after four subculture steps (96 h). However, plasmid loss could be prevented due to the presence of the TA system in pSmeLPU88b. Thus, the TA could mask the effect of the repC mutations on plasmid replication initiation and/or plasmid segregational stability. To test this hypothesis, we evaluated the bacterial growth kinetics. The assay showed that generation time was lower in the wild type (1.83±0.06 h) compared to the mutants (2.38±0.05 h for repABC mutant and 2.23±0.06 h for repC mutant. P<0,0001 Tukey’s test). The loss of pSmeLPU88b would induce the elimination of cells by the activity of TA system. These events of cell death would justify the lower growth rates observed for the mutants. These results suggest that the insertional mutations destabilize the plasmid replication, and that the TA system could be active, eliminating the cells without plasmid. Next, the stability of the two cloned replicons was separately examined in S. meliloti 2011. Plasmid pG18repABC showed similar stability to that of pSmeLPU88b (100%). In contrast, pKrepC was lost after 4 subcultures (96 h), where only 0.4% of cells retained the plasmid. Similar observations were described by Bartosik et al. [18] with the replicons of pTAVl plasmid.
To test the function of the identified parS, the plasmid pBBRparS was conjugated to LPU88 (pGrepABC) to determine if this sequence showed any incompatibility with the pGrepABC present in the strain. The analysis of the transconjugants showed that all clones only carried the pBBRparS and not the pGrepABC plasmid, confirming the incompatibility between these two elements. Therefore, this result corroborates that the identified sequence, although divergent with the consensus, is a functional parS site.
Incompatibility of each replication module to other S. meliloti plasmids
In previous work, we analyzed the incompatibility of pSmeLPU88b within a collection of rhizobia isolates, to gain insight into how this phenomenon affects the intraspecific mobilization of S. meliloti plasmids [36]. The analysis showed that four isolates, LPU57, LPU178, LPU121, and LPU122, have lost at least one plasmid in presence of pSmeLPU88b, proving that this plasmid is incompatible with some plasmids of the mentioned isolates. To investigate if some of the replication modules of pSmeLPU88b present incompatibility, plasmids pKrepC and pGrepABC were transferred to each isolate and the transconjugants were analyzed by Eckhardt gels electrophoresis. The results showed that for isolates LPU57 and LPU178 only the pGrepABC plasmid exerted incompatibility (Fig 3A). However, neither pKrepC nor pGrepABC plasmids were able to eliminate the resident plasmids in the strains LPU121 and LPU122 (Fig 3A). Perhaps, the incompatibility only manifests when both replicons are present, thus, we generated strains with plasmids pKrepC and pGrepABC. As it is shown in Fig 3B, the incorporation of both plasmids to isolates LPU121 and LPU122 did not produce any change in resident plasmids. We concluded that the incompatibility of pSmeLPU88b to the resident plasmids of strains LPU121 and LPU122 is not because of the replication modules. Thus, three options arise: both replication modules should be in the same plasmid to exert incompatibility, the incompatibility could be due to segregation issues or other genes present in pSmeLPU88b may be involved. In general, plasmid incompatibility occurs when multiple plasmids within one cell have the same replication and/or partitioning system. In the case of rhizobia, as it was mentioned before, pRetCFN42d contains, in addition to replication and partitioning genes, a gene that influences plasmid stability and incompatibility properties that is not encoded by the repABC operon [59]. In plasmids pTi-SAKURA and pTiC58 of Agrobacterium tumefaciens, two TA systems were shown to enhance plasmid stability and incompatibility [66, 67]. This could be the case of pSmeLPU88b since it carries a TA module that might compensate the loss of TAs present in the plasmids of LPU121 and LPU122 strains, but pKrepC and pGrepABC were not able to do it.
A. Profile of plasmids from isolates LPU57, LPU178, LPU121 and LPU122 before and after having received the replication modules of pSmeLPU88b. B. Plasmid profiles of isolates LPU122 and LPU121 harboring both pSmeLPU88b replication modules (plasmids pkrepC and pGrepABC).
Analysis of the replication initiator protein belonging to the repC family
Strikingly, as we mentioned before, RepC2 of plasmid pSmeLPU88b has 611 amino acids when almost all the known replication initiator proteins, RepC and RepABC families are ca. 400 amino acids. A protein alignment of RepC2 with other RepC proteins showed that the first 400 amino acids are conserved and present the typical Rep proteins’ motif. Furthermore, a BlastX analysis showed an IS4 family transposase (only c-term 44% protein identity) coded in the complementary strand of repC gene 3’-end. These results support the idea that the additional 200 C-terminal amino acids of RepC2 could be the consequence of an ancient event of an IS4 element transposition.
In order to assess if only the first 400 amino acids are enough for replication, a truncated variant of the RepC was generated by PCR, introducing a stop codon in the reverse primer and cloned in the pK18mob vector. The obtained plasmid, pKrepCT, was transferred to the strain S. meliloti 2011 to test functionality. The presence of pKrepCT was confirmed in the plasmid profiles electrophoresis (S5 Fig), confirming that the first 400 amino acids of RepC2 are able to perform replication.
Concluding remarks
Rhizobia usually carry plasmid DNA that can reach 45% of the total amount of genetic information in that cell [9]. In many cases, the information that is harbored in these replicons contributes to the saprophytic survival or to the development of symbiosis with leguminous plants. In this work, we presented the sequence of pSmeLPU88b, an accessory mobilizable plasmid of S. meliloti LPU88 [41]. The sequence analysis showed a genomic structure with backbone modules for replication/partitioning and mobilization. In addition, a region carrying information for metabolic processes that could be useful for adaptation to the environment was identified.
Plasmids must replicate within the cells. The most common replication system among rhizobial plasmids is the one based on the repABC family [13], but also repC family of replicons containing only the repC has been described in other rhizobia [33, 68]. The presence of more than one replication module is not rare in rhizobial plasmids. Interestingly, the repC family is always found in plasmids carrying a repABC family cluster. Remarkably, pSmeLPU88b showed two functional replication modules, one of them belonging to repABC and the other to repC families. In particular, repC2 of pSmeLPU88b (which belongs to the repC family) showed an atypical size, being 30% larger than the average RepC proteins. It was identified that an insertion of a mobile genetic element creates a frameshift that increase the protein size. Nevertheless, we corroborated that the first 400 amino acids are enough for the functionality of RepC2.
Despite both replicons being able to replicate, it seems that they have a different relevance. While the repABC remains stable, repC2 was lost in the same period. The low stability shown by the repC2 type of replication module in rhizobia could explain the reason why repC modules are always found in plasmids harboring a repABC module in rhizobia. In this way, the overall stability of the plasmid would be greater. Furthermore, we could speculate that repC modules are expressed in particular conditions to guarantee the prevalence of the plasmids. Moreover, the presence of two replicons could be an advantage in the environment since each replicon could have a different host range giving the plasmid a bigger chance of being horizontally transferred in the bacterial community.
Supporting information
S1 Fig. Computer-predicted secondary structure of the ctRNA.
A. Minimum free energy and centroid secondary structure of ctRNArepABC. B. Minimum free energy structure of ctRNArepC. C. Centroid secondary structure of ctRNArepC.
https://doi.org/10.1371/journal.pone.0285505.s001
(PDF)
S2 Fig. Sequence alignment of ctRNA.
The black boxes correspond to the -10 and -35 conserved promotor region; the sequence underlined corresponds to the isolated ctRNA from plasmids pRetCFN42d and pRmeGr4a; the bold G corresponds to +1 of pRetCFN42d ctRNA.
https://doi.org/10.1371/journal.pone.0285505.s002
(PDF)
S3 Fig. Plasmid profiles of S. meliloti 2011 harboring pKrepC, pGrepABC or both by Eckhardt-like gels.
https://doi.org/10.1371/journal.pone.0285505.s003
(TIFF)
S4 Fig. Plasmid profiles of S. meliloti LPU88, S. meliloti LPU88 (pKrepC) and S. meliloti LPU88 (pGrepABC) strains in Eckhardt-like gels.
The figure shows the plasmid profile of strain LPU88 before and after receiving the constructed plasmids.
https://doi.org/10.1371/journal.pone.0285505.s004
(TIFF)
S5 Fig. Plasmid profiles of S. meliloti 2011 harboring pKrepC2, pKrepC2-truncated by Eckhardt-like gels.
https://doi.org/10.1371/journal.pone.0285505.s005
(TIFF)
Acknowledgments
A.Lu. and L.G.C. are fellows of CONICET. M.F.D.P, A.La., G.T.T. and M.P are members of the Research Career of CONICET.
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