ABSTRACT
Bacterial genomes with two (or more) chromosome-like replicons are known, and these appear to be particularly frequent in alphaproteobacteria. The genome of the N2-fixing alfalfa symbiont Sinorhizobium meliloti 1021 contains a 3.7-Mb chromosome and 1.4-Mb (pSymA) and 1.7-Mb (pSymB) megaplasmids. In this study, the tRNAarg and engA genes, located on the pSymB megaplasmid, are shown to be essential for growth. These genes could be deleted from pSymB when copies were previously integrated into the chromosome. However, in the closely related strain Sinorhizobium fredii NGR234, the tRNAarg and engA genes are located on the chromosome, in a 69-kb region designated the engA-tRNAarg-rmlC region. This region includes bacA, a gene that is important for intracellular survival during host-bacterium interactions for S. meliloti and the related alphaproteobacterium Brucella abortus. The engA-tRNAarg-rmlC region lies between the kdgK and dppF2 (NGR_c24410) genes on the S. fredii chromosome. Synteny analysis showed that kdgK and dppF2 orthologues are adjacent to each other on the chromosomes of 15 sequenced strains of S. meliloti and Sinorhizobium medicae, whereas the 69-kb engA-tRNAarg-rmlC region is present on the pSymB-equivalent megaplasmids. This and other evidence strongly suggests that the engA-tRNAarg-rmlC region translocated from the chromosome to the progenitor of pSymB in an ancestor common to S. meliloti and S. medicae. To our knowledge, this work represents one of the first experimental demonstrations that essential genes are present on a megaplasmid.
INTRODUCTION
Most bacteria carry a single chromosome replicon that contains all of the genes essential for the free-living growth of the organism. Nonessential genes involved in metabolism, stress responses, and other adaptive traits are also found on chromosome and plasmid replicons. However, genome sequence analyses suggest that many bacteria carry essential genes on two replicons or chromosomes, including strains of Brucella, Burkholderia, Cupriavidus, Ralstonia, Rhodobacter, and Vibrio (1–10). In many of these organisms, there is evidence that the chromosome and chromosome replicons coevolved with a shared evolutionary history and that the chromosome replicons were initially plasmids that did not carry any essential genes. The presence of multiple replicons in certain alphaproteobacteria is well known (11, 12), and members of the Rhizobiaceae family contain a chromosome-like replicon of 3 to 4 Mb and multiple additional replicons (plasmids) that range in size from <100 kb to over 2 Mb (13–18). In the root nodule bacteria, these plasmids were initially studied because they carried nodulation (nod) and nitrogen fixation (nif) genes. In the Mesorhizobium and Bradyrhizobium genera, nod and nif genes are also known to be located in symbiotic islands on large chromosomes (>8 Mb) (19, 20). The challenges in defining autonomously replicating replicons as second chromosomes and megaplasmids have been discussed previously (21), and the term “chromid” was proposed and defined (22). We continue to use the term megaplasmids for pSymA and pSymB, since these have been used since their first identification (12).
Sinorhizobium meliloti is an alphaproteobacterium that forms root nodules on leguminous plants, including alfalfa. The bacteria differentiate into bacteroids and reduce N2 gas to ammonia within nodules. The genome of Sinorhizobium meliloti 1021 contains a 3.7-Mb chromosome and 1.4-Mb (pSymA) and 1.7-Mb (pSymB) megaplasmids (16). Symbiotic nod, nif, and fix genes are located in a 300-kb region of the pSymA megaplasmid. This plasmid carries no essential genes, as a pSymA-cured isolate grows well in laboratory media (13, 23). pSymA is a self-transmissible plasmid that can also mobilize pSymB (24). The pSymB megaplasmid has several large gene clusters involved in the biosynthesis and export of surface polysaccharides (essential for nodule invasion) and in many catabolic activities and associated solute transport systems, as well as genes that contribute to osmotic tolerance of S. meliloti (15, 25, 26). Thus, pSymB appears to play an important role in survival under diverse nutritional conditions present in the soil and rhizospheres (21).
The G+C content, dinucleotide composition, codon usage, and distribution of intergenic mosaic elements suggest that pSymB and the chromosome of S. meliloti 1021 share the same evolutionary history, whereas pSymA appears to have been acquired more recently through horizontal transfer (15, 16, 27–29). Bioinformatic analysis of the pSymB sequence identified an ArgtRNACCG sequence; however, other than the minCDE genes, which were subsequently shown not to be required for cell viability (30), other possibly essential genes were not identified. Here we demonstrate experimentally that the engA and tRNAarg genes on pSymB are essential for cell viability. To allow the deletion of this region from the pSymB megaplasmid, we describe the construction of strains in which the engA and tRNAarg genes are stably integrated into the chromosome. Phylogenetic and gene synteny data are presented that strongly suggest that a 69-kb region containing these genes translocated from the chromosome to a progenitor of the present pSymB megaplasmid. These findings represent a clear example of genes on a megaplasmid or second chromosome being determined experimentally to be essential. Moreover, the evidence supporting the chromosomal origin of these genes in an ancestral strain appears to be particularly clear.
MATERIALS AND METHODS
Media, growth conditions, and bacterial strains.Luria-Bertani (LB) broth, M9 medium, bacterial matings, ΦM12 transduction, and general manipulations were used or performed as previously described (31). Bacterial strains are described below and in Table 1, and the sequences of oligonucleotides used in this study are listed in Table S1 in the supplemental material. Antibiotic (streptomycin [Sm], neomycin [Nm], spectinomycin [Sp], gentamicin [Gm], tetracycline [Tc], and chloramphenicol [Cm]) concentrations in liquid and solid media were as previously described (26, 31). For construction of strains carrying Flp recombinase target (FRT) sites flanking pSymB regions, one FRT site was from the pTH1522 (Gmr FRT) insertion library (31), and the second was obtained by cloning target pSymB fragments into the suicide plasmid pTH1937 (Nmr FRT). The pTH1937 derivatives were recombined into the pTH1522 pSymB integrants. Flp recombinase was transferred to the FRT strains on the Tcr plasmid pTH1944, which replicates in S. meliloti (32).
Bacterial strains and plasmids
Analyses of engA and tRNAarg genes.The engA gene is 1,428 nucleotides (nt) long. To analyze engA and the surrounding genes, various PCR-amplified fragments were cloned into the suicide plasmid pTH1703, which replicates in Escherichia coli but not in S. meliloti (31). Plasmid pTH1996 contains a 2.1-kb NsiI/BglII PCR fragment (amplified using primers ML11667 and ML11660), from nt 185 of engA to nt 869 downstream of the engA stop codon, cloned into pTH1703. Plasmid pTH1997 contains a 740-bp XhoI/BglII fragment (amplified using primers ML11659 and ML11660) internal to engA, from nt 185 to 925, in pTH1703. Plasmid pTH2544 carries a fragment upstream of engA (amplified using primers ML09-2099 and ML09-2100) cloned into the BglII-XhoI sites in pTH1703. Plasmid pTH2608 carries the 431-bp intergenic region (amplified using primers ML09-2098 and ML09-2097) between smb20996 and smb20997 cloned into the BglII-XhoI sites of pTH1703. Plasmids pTH2609 and pTH2607 carry internal fragments of smb20996 (609 bp; amplified using primers ML09-2096 and ML09-2097) and smb20997 (249 bp; amplified using primers ML09-2106 and ML09-2107), respectively, cloned into the BglII-XhoI sites in pTH1703. Plasmids pFL2987 (nt 1,233,633 to 1,235,118) and pFL5726 (nt 1,233,020 to 1,235,022) were from the S. meliloti gene fusion library in pTH1522 (pTH1703 is derived from pTH1522) (31).
For complementation experiments, the complete engA coding region (amplified using primers ML11753 and ML11754) was cloned as a PacI fragment into the broad-host-range expression vector pTH1931 (Table 1) to generate the plasmid pTH2027. A 460-bp PacI fragment (amplified using primers ML20184 and ML20185) from 375 nt upstream of the complete tRNAarg gene (smb21712; 77 nt long) was amplified by PCR and ligated into the expression vector pTH1931 to produce plasmid pTH2302. Plasmids pTH2302 and pTH2027 were used to provide the engA and tRNAarg genes, respectively, in trans.
Construction of the attP delivery vector pTH2602.A ϕC31 attP site (33), prepared by annealing the complementary oligonucleotides attP1 and attP2, was cloned into the XhoI and KpnI sites of pUX19 (34) to give pTH2596. The Spr Smr Ω cassette (35) was cloned as a KpnI/EcoRI fragment into pTH2596 to give pTH2599. A second attP site, prepared by annealing oligonucleotides attP3 and attP4, was cloned into EcoRV/XbaI-digested pUX19 to give pTH2582. The attP site and Spr Smr cassette were excised from pTH2599 with XhoI and EcoRI and then cloned into pTH2582 to give pTH2602. Only one attP site is required for integration, and the second was inserted to allow manipulations that are not described in this report.
Construction of the engA and tRNAarg chromosomal integration vector pTH2750.A 320-bp PstI/BamHI fragment containing the tRNAarg gene and its promoter (pSymB nt 1,315,000 to 1,315,301) was PCR amplified (using primers ML09-1896 and ML09-1897) and ligated into pTH2602 to give pTH2604. A 2,475-bp PmlI/NsiI fragment (pSymB nt 1,233,512 to 1,231,037) from 215 nt upstream of smb20996 to 38 nt downstream of engA was PCR amplified (using primers ML10-3867 and ML10-3868) and ligated into pTH2604 to give pTH2750. A pTH2602 derivative, pTH2763, which carries smb20996 and engA alone (without the tRNAarg gene), was also constructed.
Construction of S. meliloti strain carrying attB and the ϕC31 integrase.An 881-nt fragment from the S. meliloti chromosome (nt 3,233,592 to 3,234,473) with a central SmaI site was PCR amplified using primers ML19617 and ML19618 and cloned into the NotI site of the Gmr suicide plasmid pJQ200ucI (36) to give pTH2269. The ϕC31 attB site, prepared by annealing the oligonucleotides ML3818 and ML3819, was cloned into the SmaI site to give plasmid pTH2287. Plasmid pTH2287 was transferred to S. meliloti RmP110, and single-crossover recombinants, purified on LB agar with Gm and Sm (LB Gm Sm), were grown in LBmc (LB, 2.5 mM CaCl2, 2.5 mM MgSO4) and plated on LB agar containing Sm and 5% sucrose to select for loss of the sacB gene present in pTH2287. Recombinants were examined by PCR using primers ML07-1019 and ML07-1020, which annealed outside the originally amplified intergenic region. The resulting fragment was digested with StyI, which cut the fragment only if the attB site was present. The sequence of the amplified DNA for one attB recombinant was confirmed, and the strain was designated S. meliloti RmP1683. We note that attB was integrated 5 kb from the metH gene and that this allows unmarked attB or attB integrants to be transduced easily to metH auxotrophic strains.
To insert the ϕC31 integrase into S. meliloti, the chromosomal region from nucleotides 3,417,143 to 3,418,489 was PCR amplified using primers ML20437 and ML20438 and cloned into the NotI site of pJQ200ucI. The resulting plasmid, pTH2339, was digested with SacI (cuts a single central site in the S. meliloti insert DNA), and then a 6-kb integrase cassette excised as a SacI fragment from pTH1707 was cloned into the SacI site to give plasmid pTH2404. Plasmid pTH2404 was transferred to S. meliloti RmP1683, and single-crossover recombinants, purified on LB Gm Sm plates, were grown in LBmc and plated on LB agar containing streptomycin and 5% sucrose. Colonies from these plates were examined for the presence of the ϕC31 integrase and the loss of pTH2404 by a PCR using one primer (ML07-244) that annealed outside the originally amplified intergenic region and one that annealed within the integrase gene (ML07-1191). The presence of a 3.8-kb fragment confirmed the integration of the integrase, and the resulting strain was designated RmP1685. The 6-kb ϕC31 integrase cassette in S. meliloti RmP1685 carries a neomycin resistance (Nmr) gene flanked by 2 FRT sites. To delete the Nmr gene, the Tcr pTH1944 plasmid carrying flp (32) was transferred to RmP1685. A Tcr Nms transconjugant was purified and designated S. meliloti RmP2667.
Integration of tRNAarg and engA genes into the chromosome and their deletion from pSymB.To integrate the engA and tRNAarg genes into the S. meliloti chromosomal attB site, plasmids pTH2602, pTH2750 (smb20996-engA-tRNAarg), pTH2604 (tRNAarg), and pTH2763 (smb20996-engA) were transferred to RmP2667, and Spr Smr recombinants were selected on M9 medium with 5 mM l-4-hydroxyproline (on which E. coli cannot grow) and purified. Phage lysates of these strains were used to transduce the integrated constructs into a metH::Tn5-132 auxotroph. Ninety-five percent of Spr transductants were Met prototrophs, as expected if the integration occurred at attB as opposed to the pSymB smb20996, engA, and tRNAarg genes. The structure of the integrated constructs was verified by PCR using primers ML11-3174 and ML11-3175, which flank the attB site. The absence of a 482-bp band was consistent with integration of the plasmid at the attB site.
Sequence analyses.Multiple sequence alignments were performed using ClustalW2 (37, 38). Genomes were aligned with the open-source MAUVE aligner, version 2.3.1, using the progressive algorithm, iterative refinement, a seed weight of 15, and sum-of-pairs local colinear block scoring (39). Dot plots were generated with YASS (40).
Genome sequences were downloaded from GenBank, and the corresponding accession numbers are as follows: AL591688.1 (S. meliloti 1021 chromosome) (41), AL591985.1 (S. meliloti 1021/pSymB) (15), CP001832.1 (S. meliloti SM11/pSmeSM11d) (42), CP002782.1 and CP002742.1 (S. meliloti AK83 chromosome 2 and S. meliloti BL225C/pSINMEB02) (43), CP000738.1 and CP000739.1 (Sinorhizobium medicae WSM419 chromosome and pSMED01) (44), CP001389.1 and CP000874.1 (Sinorhizobium fredii NGR234 chromosome and pNGR234b) (45), NC_016812.1 (S. fredii HH103 chromosome) (46), and CP003563.1 (S. fredii USDA 257) (47).
RESULTS
The pSymB engA gene is essential for growth of S. meliloti.The pSymB megaplasmid is 1,683,333 bp long, and nucleotide position 1 was assigned as the first nucleotide of the lacE gene (15). We are using the FRT/Flp recombination system (48) to delete segments of the S. meliloti genome (B. Milunovic and T. M. Finan, unpublished data). In this procedure, FRT sites are inserted in direct orientation at the ends of the region to be deleted. Transfer of a plasmid expressing Flp recombinase (pTH1944; Tcr) then results in recombination between FRT sites and in deletion of the FRT site-flanked region. The recovery of recombinants upon transfer of Flp recombinase (transfer frequency, ∼10−3/recipient) suggests that the FRT site-flanked region carries no genes required for cell viability, whereas the inability to recover recombinants upon the transfer of Flp recombinase suggests that loci in the FRT site-flanked region are required for cell viability.
FRT site-directed deletions were recovered for most of pSymB; however, no FRT site recombinants were obtained for strains in which FRT sites flanked the 52-kb B110 region (nt 1,207,052 to 1,255,032), the B115 region (nt 1,207,052 to 1,243,616), or the B113 region (nt 1,207,052 to 1,233,633) (Fig. 1). However, recombinants were recovered which had lost the FRT site-flanked region B111 (nt 1,207,052 to 1,223,871). These data suggested that an “essential” locus lay in the 9.8-kb region from nt 1,223,871 to 1,233,633. This region carries the smb20991, smb20992, smb21674, smb20993, smb20994, engA, smb20996, and smb20997 genes (Fig. 1). In particular, the 1,431-bp engA gene encodes a member of the Der subfamily of bacterial GTPases, which have been found to be essential in bacteria such as E. coli and Bacillus subtilis (49, 50).
(A) Linear representation of the pSymB megaplasmid showing the locations of engA and other notable genes in the surrounding region. FRT site-flanked regions are indicated with two-sided arrows, and the ability (+) or inability (−) to recover deletions is indicated. The dotted line indicates the B179 FRT site-flanked region, which includes the engA and tRNAarg gene region. (B) Map showing engA and the surrounding genes on pSymB. DNA fragments cloned into the suicide vector pTH1703 are represented by bold lines, and the ability (+) or inability (−) to recover S. meliloti recombinants is indicated.
To investigate whether engA is essential for S. meliloti cell viability, we attempted to construct an engA null allele by single-crossover plasmid integration. Various DNA fragments from the engA gene region were cloned into the suicide plasmid pTH1703, as illustrated in Fig. 1 and described in Materials and Methods. The resultant plasmids were transferred by conjugation to S. meliloti, and the recovery of plasmid recombinants (+) or no recombinants (−) is indicated following the plasmid name in Fig. 1. Plasmid pTH1997 carried a 740-bp fragment internal to the engA gene, while pTH1996 carried a fragment from nt 185 of the engA gene to 869 nt downstream of the engA stop codon. No S. meliloti Gmr transconjugants were recovered for pTH1997 (<10−8/recipient), whereas Gmr transconjugants were readily recovered for pTH1996, pFL2987, and pFL5726 (∼10−4 to 10−5/recipient). Because pTH1997 recombinants would be engA mutants, whereas pTH1996 recombinants would be engA+ strains, these data suggested that the disruption of engA is lethal. The absence of pTH2544 and pTH2609 recombinants suggested that smb20996 and engA are cotranscribed from a promoter that lies upstream of smb20996, and the data are ambiguous as to whether smb20996 is an essential gene. The recovery of pTH2607 recombinants showed that smb20997 is not required for cell viability and that the smb20996-engA promoter is downstream of smb20997. Microarray transcriptome data for the S. meliloti genome indicate that smb20996 and engA are transcribed at similar levels (see the supplemental data for reference 51), and analysis of directional RNA sequence reads from this region indicates the presence of a single transcriptional start site upstream of smb20996 (see Fig. S1 in the supplemental material).
For complementation experiments, engA alone was cloned downstream of the lac promoter in the broad-host-range Spr plasmid pTH1931, giving pTH2027. When pTH2027 was present in the FRT site-flanked B110 (RmP734) and B115 (RmP826) strains, FRT site recombinants were readily recovered upon transfer of the Flp recombinase plasmid pTH1944. This result demonstrated that engA is essential and that it is the only essential gene in the B110 region of the pSymB megaplasmid. The generation of the ΔB110 and ΔB115 deletions in the strains carrying engA in trans on pTH2027 was confirmed by PCR using four sets of primers that amplified fragments from within and outside the B110 region (strain RmP734; primers 2626UF-UR, 2626DF-DR, 4440UF-UR, and 4440DF-DR) and the B115 region (strain RmP826; primers 641UF-UR, 641DF-DR, 4440UF-UR, and 4440DF-DR) (see Table S1 in the supplemental material).
The unique copy of the tRNAarg gene on pSymB is essential for S. meliloti.No FRT site recombinants were obtained for the FRT site-flanked B117 region (nt 1,308,912 to 1,528,150), whereas recombinants were readily obtained for strains in which FRT sites flanked the B118 region (nt 1,323,078 to 1,528,150) (Fig. 2). This suggested that an essential gene may lie in the 14.2-kb region from nt 1,308,912 to 1,323,078 (Fig. 2). Consistent with this suggestion, no FRT site deletions were recovered for the B158 region (nt 1,308,912 to 1,322,226). This 13-kb region carries the unique copy of a tRNA gene specific for CGG, and this was previously suggested to be an essential gene (15).
Linear representation of the pSymB megaplasmid showing the locations of the tRNAarg gene and other notable genes in the surrounding region. FRT site-flanked regions are indicated with two-sided arrows, and the ability (+) or inability (−) to recover deletions is indicated.
To investigate whether the tRNAarg gene was essential and was the only essential gene in the 14-kb region, the 77-nt tRNAarg gene (smb21712) and its upstream promoter region were cloned as a 459-bp fragment in a replicating plasmid (pTH2302), and this plasmid was transferred to strain RmP896, in which the B158 region is flanked by FRT sites. ΔB158 FRT site recombinants were readily obtained when the parent B158 strain contained the pTH2302 plasmid, whereas no FRT site recombinants were obtained in the absence of pTH2302. We concluded that the only essential gene in the B158 region (nt 1,308,912 to 1,322,226) is the tRNAarg gene. The absence of the B158 region in ΔB158 FRT site recombinants (e.g., RmP896 carrying pTH2302 tRNAarg in trans) was confirmed by PCR using four sets of primers that amplified fragments from within and outside the B158 region (primers 3486UF-UR, 3486DF-DR, 422UF-UR, and 422DF-DR; see Table S1 in the supplemental material).
Integration of tRNAarg and engA genes into the chromosome and their deletion from pSymB.Having established that the engA and tRNAarg genes are required for cell viability, we wished to integrate these genes into the S. meliloti chromosome and to delete the region containing these genes from the pSymB megaplasmid. To accomplish this, we adapted the Streptomyces coelicolor attB/attP phage ϕC31 integration system for use in S. meliloti. Phage ϕC31 integrase catalyzes site-specific recombination between the attP site of ϕC31 and the attB site in the S. coelicolor chromosome (33). An Spr attP suicide plasmid, pTH2602, as well as derivatives that carried the smb20996-engA and tRNAarg genes (pTH2750) or only the tRNAarg gene (pTH2604) or smb20996-engA (pTH2763), were integrated into a ϕC31 attB site in the chromosome of S. meliloti strain RmP2667, which also carried the ϕC31 integrase gene (Fig. 3). The locations and structures of the integrated constructs in the chromosome were verified by transduction and PCR as outlined in Materials and Methods.
Schematic illustration of the integration of the tRNAarg and smb20996-engA genes into the S. meliloti chromosome. Plasmid pTH2750, carrying the tRNAarg gene and smb20996-engA, was transferred into S. meliloti RmP2667, which expresses ϕC31 integrase and carries a ϕC31 attB site in the chromosome. Integrase catalyzes recombination between the attB site in the chromosome and one of the attP sites on the plasmid. Site-specific recombination of attB and attP generates the attL and attR sites, which are not recognized by the integrase. The illustration also includes the metH gene, which can be used for transfer of the attB site or integrants at the attB site via transduction into a metH mutant strain.
To determine whether the chromosomal integration of the engA and tRNAarg genes would allow the deletion of these genes from pSymB, the pTH2602-derived insertions in the chromosome were transduced via Spr selection into S. meliloti strain RmP2677, which carries FRT sites flanking a 115-kb region, designated B179, that includes the engA and tRNAarg genes (flanked by FRT sites; nt 1,207,052 to 1,322,226) (Fig. 1 and 2). Upon transfer of the Flp plasmid pTH1944, the ΔB179 recombinants were recovered only for S. meliloti strain RmP2694, in which both smb20996-engA and the tRNAarg gene were integrated into the chromosome. RmP2694 transconjugants were recovered at a frequency of ∼10−4, and a representative of S. meliloti RmP2694 ΔB179 was purified and designated S. meliloti RmP2712. The ΔB179 deletion in this strain is marked by an Nmr Gmr phenotype and can be transduced by selecting for Nmr or Gmr recombinants. When the ΔB179 strain was transduced into the various pTH2602 derivatives of wild-type RmP110 (i.e., RmP2683, RmP2684, RmP2685, and RmP2686), no transductants were recovered (frequency of <10−9), except for when S. meliloti RmP2686 (RmP110 attB::pTH2750) was the recipient (frequency of ∼10−7). These results show that the chromosomal integration of smb20996-engA and the tRNAarg gene allows the deletion of these genes from pSymB, and together, the data confirm that the engA and tRNAarg genes are the only essential genes in the B179 region.
Mapping of previously constructed pSymB deletions.Deletions of pSymB were previously isolated by screening for recombination between the IS50 elements of Tn5 derivatives that flanked various regions (52). No deletions were recovered for three distinct regions. To determine the relationship between those deletions and the engA and tRNAarg genes and to align the previous deletion map with the DNA sequence of pSymB, we determined the nucleotide sequence of the IS50-pSymB junctions for 34 transposon insertions used in constructing the previous pSymB genetic map (see Fig. S2 in the supplemental material). The data showed three regions for which deletions were not recovered. The first region, from Ω5085 (nt 106,128) to Ω5079 (nt 26,409), included the repABC genes and pSymB oriV, which are essential for megaplasmid replication (53). Deletions of this region could not be recovered because pSymB carries essential genes. The second region, a 35-kb region from Ω5047 (nt 735,511) to Ω5060 (nt 770,089), has since been deleted (unpublished data), and the third region, a 275-kb region from Ω5011 (nt 1,177,742) to Ω5177 (nt 1,452,882), included the engA and tRNAarg genes described here.
Origin of the engA and tRNAarg genes.In considering the ancestry of the engA and tRNAarg genes, we observed that when the genomic location of engA and tRNAarg homologs was superimposed on a 16S rRNA gene phylogenetic tree of members of the Rhizobiaceae (Fig. 4), the tree showed that both the engA and tRNAarg genes were located on the main chromosome, except in the case of all sequenced S. meliloti strains (15 strains) and S. medicae WSM419 (16, 43, 44, 54, 55). Furthermore, both the engA and tRNAarg genes were located on the chromosome for Sinorhizobium fredii strains NGR234, USDA257, and HH103 (45–47) and nine recently sequenced Sinorhizobium strains (seven S. fredii strains, one Sinorhizobium sojae strain, and one Sinorhizobium sp. strain [56]). Since S. meliloti and S. medicae form a monophyletic group, we hypothesized that the engA and tRNAarg genes were chromosomally encoded in an early Rhizobiaceae ancestor and that following the divergence of S. meliloti from S. fredii, but before the split from S. medicae, these genes were transferred to the precursor of the pSymB megaplasmid.
Phylogenetic tree of the Rhizobiaceae family based upon 16S rRNA gene sequences, created using the weighted neighbor-joining method of the Ribosomal Database Project (80). E. coli was used as an outgroup. The location of the engA and tRNAarg genes in each species is indicated on the right. Only S. meliloti and S. medicae carry both genes on a megaplasmid, while both are carried by the main chromosome in the other species.
The translocated engA-tRNAarg-rmlC region.Alignment of the engA-tRNAarg regions from the S. fredii NGR234 chromosome, the S. medicae WSM419 pSMED01 megaplasmid, and the S. meliloti 1021 pSymB megaplasmid by use of progressive MAUVE (39) (see Fig. S3A in the supplemental material) showed that almost an entire 69-kb region of S. fredii was present on the pSymB and pSMED01 megaplasmids, although several insertions were apparent for the S. meliloti 1021 region. The region of synteny was delimited by the engA gene at one end and the rmlC gene at the other boundary. We refer to this genome segment as the engA-tRNAarg-rmlC region. A DNA dot plot of the engA-tRNAarg-rmlC region, including 20 kb on each side from the S. meliloti pSymB plasmid and the S. fredii chromosome, allowed easy visualization of the synteny in this region (Fig. 5B). Virtually the entire 69-kb engA-tRNAarg-rmlC region from S. fredii was present within the 129-kb engA-tRNAarg-rmlC region in S. meliloti. Most of the size difference resulted from two ∼25-kb insertions and one 8-kb insertion in S. meliloti. These segments appear to correspond to insertions in S. meliloti rather than deletions in S. fredii, as the corresponding engA-tRNAarg-rmlC region in S. medicae has only one extra segment (Fig. 5D), and although it is in the same position as an insertion in S. meliloti (beside the tRNAarg gene), the inserted sequence is not conserved (Fig. 5A). It is more likely for three additional segments to be inserted in this region in S. meliloti than it is for all three to be deleted from S. medicae and S. fredii independently.
Dot plots showing alignments of the S. meliloti 1021 and S. medicae WSM419 engA-tRNAarg-rmlC regions (A), the S. meliloti and S. fredii NGR234 engA-tRNAarg-rmlC regions (B), the S. meliloti engA-tRNAarg-rmlC region and the S. fredii translocation target site (C), the S. medicae and S. fredii engA-tRNAarg-rmlC regions (D), and the S. fredii engA-tRNAarg-rmlC region and S. meliloti translocation donor site (E). The plots were generated with YASS (48), and for each plot, a 20-kb sequence from each side of the region of interest was included.
Donor and target sites in S. fredii, S. meliloti, and S. medicae.In S. fredii NGR234, the engA-tRNAarg-rmlC region in the chromosome is flanked on one side by kdgK, which lies 151 nucleotides from engA. On the other side, a dppF2 ortholog (NGR_c24410) is located 252 nt from rmlC. This is the first gene of a six-gene cluster (NGR_c24410 to NGR_c24460) that encodes a putative oligopeptide transport system. In S. medicae WSM419, kdgK and the ortholog of dppF2 are 50 nt apart, while in the S. meliloti 1021 chromosome, an insertion element (ISRm5; 1,096 nt) is located in the 1,674-nt region between kdgK and dppF2. Thus, the whole of the 69-kb engA-tRNAarg-rmlC region has been removed from the S. medicae and S. meliloti chromosomes (Fig. 5E; see Fig. S3B in the supplemental material).
To identify the target site into which the 69-kb engA-tRNAarg-rmlC region inserted in the ancestral pSymB replicon, we searched the 2.43-Mb S. fredii pNGR234b megaplasmid for synteny to the pSymB region flanking engA-tRNAarg-rmlC, and a putative target region was identified (see Fig. S3C in the supplemental material). As can be seen in a dot plot of the corresponding regions of pSymB and pNGR234b, a 36-kb region from pNGR234b is conserved on pSymB and flanks the engA-tRNAarg-rmlC region (Fig. 5C). The pNGR234b region from nt 1,083,197 to 1,069,822 shows conservation of the pSymB sequence from nt 1,359,859 to 1,374,631, starting at smb21429, the gene directly downstream of rmlC. On the other side, the pNGR234b region from nt 1,102,607 to 1,090,470 aligns with the pSymB region from nt 1,215,001 to 1,225,235, ending in the intergenic region following nfeD, which is the sixth gene before engA. The five genes between nfeD and engA are also present in the same location on pSMED01 of S. medicae WSM419 but are absent from pNGR234b. Additionally, the region from nt 1,083,184 to 1,090,477 on pNGR234b carries six genes encoding a probable ABC-type sugar transport system that is absent from pSymB and pSMED01. A dot plot of the whole of NGR234 pNGR234b and S. meliloti 1021 pSymB revealed considerable synteny, but many rearrangements can be seen (see Fig. S4 in the supplemental material).
Nucleotide sequence alignments were used to examine the borders of the engA-tRNAarg-rmlC region more closely. Sequences from 100 nt upstream and downstream of the last nucleotides of engA and rmlC from four S. meliloti (1021, AK83, BL225C, and SM11), one S. medicae (WSM419), and three S. fredii (NGR234, HH103, and USDA257) strains were included. The engA alignment (see Fig. S5A in the supplemental material) revealed a high level of conservation between all strains until the engA stop codon, which is UGA in all S. meliloti and S. medicae strains but varies among S. fredii strains. The 100 nucleotides downstream of engA are largely conserved between S. meliloti strains, but less so between S. meliloti and the other species. Examination of the rmlC alignment (see Fig. S5B and S6) revealed that the rmlC coding regions in S. meliloti 1021 and S. fredii NGR234 are well conserved (82%), while the corresponding region in S. medicae appears to have degenerated and carries insertions/deletions. The sequence similarity between S. meliloti, S. medicae, and S. fredii ends a few nucleotides before the rmlC stop codon (see Fig. S5B). These data suggest that the end of synteny for the engA-tRNAarg-rmlC region between these species occurs right at the end of engA and rmlC.
DISCUSSION
We have presented experimental evidence that the engA and tRNAarg genes located on the pSymB megaplasmid are essential for growth of S. meliloti. The tRNAarg locus of pSymB was previously suggested to be essential for cell viability, and it was also suggested that the tRNAarg gene had a chromosomal origin (15, 16). S. meliloti contains four tRNAarg species for the six arginine codons. Three tRNAarg species, with the anticodons CCU, UCU, and ACG, are chromosomally encoded, and the ACG anticodon can recognize three codons (CGU, CGC, and CGA) through modification of the adenine to inosine and via wobble base pairing. However, the chromosomally encoded arginine tRNAs are unable to pair with CGG, which is the second most frequently used codon for arginine in the S. meliloti genome (27). The pSymB tRNAarg gene encodes the only tRNA specific for the CGG codon, and as such, it is expected to be an essential gene.
The identification of engA as an essential gene in S. meliloti is entirely consistent with reports showing that it is essential in a variety of Gram-positive and Gram-negative organisms, such as E. coli and B. subtilis (49, 50, 57, 58). EngA is a broadly conserved bacterial GTPase with two GTP binding domains and is likely involved in ribosome biogenesis (59). S. meliloti engA is located 122 nt downstream of smb20996, and results from plasmid integration experiments strongly suggest that engA is transcribed from a promoter that lies upstream of smb20996 (Fig. 1). The S. fredii NGR234 smb20996 ortholog, NGR_c23740, is only 11 nt upstream of engA, and hence, in S. fredii, it is also likely that engA is transcribed from a promoter upstream of NGR_c23740. The smb20996 gene is not essential for cell viability, as expression of engA alone was sufficient to complement deletion of both smb20996 and engA in the ΔB110 and ΔB115 strains (Fig. 1).
The high level of gene synteny between the engA-tRNAarg-rmlC regions in S. meliloti pSymB, S. medicae pSMED01, and the S. fredii chromosome strongly suggests that the whole 69-kb region translocated from the chromosome to the megaplasmid in a single translocation event. A schematic representation of the proposed history of the engA-tRNAarg-rmlC region is shown in Fig. 6. The evidence suggests that the translocation of the engA-tRNAarg-rmlC region occurred in an ancestral cell (Fig. 6, panel III) and that the mechanism of translocation and integration into pSymB likely resulted in its simultaneous loss from the chromosome. Alternatively, one could envisage a translocation in which the chromosome retained a copy of the translocated region and then subsequent purifying selection via deletion events resulted in the loss of the duplicated region. The latter appears unlikely, as alignments showed a precise loss of this region from the S. meliloti and S. medicae chromosomes and no remnant fragments that could indicate that a progressive decay was present (Fig. 5E; see Fig. S3B in the supplemental material). Moreover, the genes that flank this region in S. fredii are located beside each other in S. medicae, and an ISRm5 element is located between these genes in the S. meliloti sequence (Fig. 6, panels III, IV, and V). It has been suggested that pSymB originated as a nonessential megaplasmid that has been present in the S. meliloti lineage since before its divergence from Agrobacterium tumefaciens (15, 28, 29). In this scenario, none of the genes that originated with pSymB should be required for viability, unless S. meliloti evolved to become dependent upon one or more of them (17). In the case of the engA and tRNAarg genes, the latter possibility can be eliminated because these genes appear to be essential for cell viability in all bacteria.
Schematic representation of the chromosome and pSymB-like replicon for five extant and extinct members of the Sinorhizobium genus, showing the proposed history of the 69-kb engA-tRNAarg-rmlC region (hatched region). Roman numerals are used to relate each chromosome and pSymB-like pair to the appropriate node or internode.
The region flanking the engA-tRNAarg-rmlC region in S. fredii is present in a contiguous segment in the S. meliloti chromosome, separated by an insertion element, while the region flanking the engA-tRNAarg-rmlC region on pSymB is present as a contiguous fragment on the pNGR234b plasmid of S. fredii, separated by a probable ABC-type sugar transport system (Fig. 6). Examination of the regions directly flanking the engA-tRNAarg-rmlC region in S. meliloti 1021 did not reveal any obvious features that may have facilitated the translocation, other than the insertion element ISRm5, located between kdgK and dppF2 in S. meliloti.
It is also curious that the ancestral pSymB plasmid carrying the engA-tRNAarg-rmlC region was fixed in the S. meliloti and S. medicae lineage. Perhaps this was simply the fixation of a random event, or perhaps the translocation of the engA-tRNAarg-rmlC region to the megaplasmid resulted in a fitness advantage. In this respect, it is interesting that in addition to the two essential genes, the translocated region contains a significant number of other genes that are noteworthy with respect to plant-microbe interactions (Fig. 1 and 2). The bacA gene lies 5 kb from engA and is essential for bacteroid development in S. meliloti. It is important for prolonged intracellular survival during host-pathogen interactions, as in chronic infection by the alphaproteobacterium Brucella abortus (60–63). The bacA gene is located 2 kb downstream of engA on the main chromosome of Brucella abortus strain 9-941. Of seven genes (gbpR and araABCDEF) involved in arabinose utilization (64), araA encodes a predicted solute-binding protein for arabinose. This is an ortholog of the chvE gene, which has been studied because of its role in attenuating virulence in Agrobacterium tumefaciens (64–66). chvE (araA) is ∼6.5 kb and ∼10 kb from the tRNAarg gene in A. tumefaciens and S. meliloti, respectively. The idhA gene is required for utilization of myo-inositol in S. meliloti and S. fredii (67, 68), and in S. fredii NGR234, the idhA2 gene lies between the tRNAarg gene and chvE (araA) on the chromosome. A functional myo-inositol dehydrogenase gene is required for nodulation competition in S. meliloti (68) and for S. fredii USDA191 to efficiently fix N2 and competitively nodulate soybean plants (69). Other genes in the engA-tRNAarg-rmlC region that are less associated with plant-microbe interactions include bdhA, glpK, and xdhA, involved in the utilization of 3-hydroxybutyrate, glycerol, and purines, respectively (70, 71).
Among microbial species that carry essential genes on more than one replicon, there is one major chromosome carrying most of the essential genes and a second replicon that has just a few genes predicted to be essential (72). Various reports have suggested that the second replicons obtained their essential genes from the main chromosome. In a recent report, a 180-kb conjugative plasmid from a bean-nodulating Sinorhizobium fredii strain was shown to contain a 40-kb region with high similarity to a region from the S. fredii NGR234 chromosome. The genes carried in this region appear to be involved in small-molecule metabolism and are not essential (14). Other examples in which essential genes appear to have been transferred to second replicons include p42e of Rhizobium etli (73), pNRC100 of Halobacterium sp. strain NRC-1 (74), chromosome III of Burkholderia cenocepacia AU-1054 (4), pSmeSM11d of S. meliloti SM11 (42), the linear chromosome of A. tumefaciens (17), and the second chromosome of Vibrio cholerae (5, 75). However, we are aware of few studies in which postulated essential genes on second replicons have been verified experimentally. In two such analyses, the RHE_PE00001 and RHE_PE00024 genes of the 505-kb p42e replicon of Rhizobium etli were shown to be essential for growth in rich medium (73), and the orc2 gene, located on the 365-kb pNRC200 replicon of Halobacterium sp. NRC-1, was shown to be required for cell viability and may play a role in DNA replication (76). We believe there is a need to experimentally verify the presence of essential genes on second replicons, as our knowledge of what constitutes an essential gene complement is incomplete and such studies could uncover functional redundancies. Additionally, our reliance on bioinformatics to identify essential genes on secondary chromosomes may result in an overestimation of the percentage of bacteria that carry essential genes on multiple replicons. As such, experimental verification could contribute to our understanding of prokaryotic genome organization. However, the large and increasing number of genome sequences precludes such functional studies in all but a few organisms.
The Sinorhizobium genus is expanding as new root nodule isolates are identified from diverse leguminous plants and geographic regions (14, 43, 56, 77–79). It will be interesting to see how the chromosome or megaplasmid location of the engA and tRNAarg genes segregates in these newly identified Sinorhizobium species. In the case of S. meliloti, the presence of both the engA and tRNAarg genes on pSymB explains why attempts to cure S. meliloti of pSymB have been unsuccessful (52, 53). An S. meliloti derivative cured for pSymA has been obtained (23), and the integration of the engA and tRNAarg genes into the S. meliloti chromosome may now allow the curing of pSymB, and perhaps the construction of a strain in which both plasmids have been removed. S. meliloti strains lacking pSymB, and possibly both pSymA and pSymB, could be valuable for genomic studies, including the identification of minimal symbiotic gene sets and the identification of genetic requirements for multiple phenotypic traits.
ACKNOWLEDGMENTS
This work was supported by grants to T.M.F. from the Natural Sciences and Engineering Research Council of Canada. G.D. was supported in part by NSERC USRA and by an Ontario Genomics Institute (OGI) summer research fellowship. T.M.F. also acknowledges support from Genome Canada through the OGI and the Ontario Research and Development Challenge Fund.
We are very grateful to R. Morton and B. Golding for assistance, interest, and advice, to C. Patten and Adrea Fink for assistance with ϕC31 integrase experiments, and to Susan Lehman for mapping of pSymB Tn5 insertions.
FOOTNOTES
- Received 19 September 2012.
- Accepted 25 October 2012.
- Accepted manuscript posted online 2 November 2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01758-12.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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