oriT-directed cloning of defined large regions from bacterial genomes: identification of the Sinorhizobium meliloti pExo megaplasmid replicator region

Abstract

We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.

FIG. 1

Illustration of integration plasmids pTH455 and pTH509. The IS50-oriV-ΩSp-oriT integration cassettes in the two plasmid backbones (pBluescript and pBACe3.6) differ in the orientation of the IS50 fragment. The IS50 is shaded white (outside end) to black (inside end). All indicated restriction sites are unique except for the SmaI and SacI sites indicated in pTH509. E, EcoRI; I, I-SceI; K, KpnI; Sc, SacI; SI, SalI; Sm, SmaI; Sp, SpeI.

FIG. 2

Schematic representation of predicted oriT-directed transfer events during conjugation from E. coli(pTH504) to either E. coli (trfA) or S. meliloti. Transfer is initiated by nicking at the oriT nic site and occurs 5′ to 3′. The 5′ end is thought to remain covalently attached at the cell membrane and is then ligated to the 3′ end of an identical nic site (31, 41, 51). Two such nicking-ligation events can occur in pTH504, resulting in the transfer of two distinct regions (the two types of dashed lines) originating from either oriT site. Rescue of the IS50-oriV-ΩSp-oriT cassette plasmid (pTH582) via IS50-directed recombination in S. meliloti at Ω5069::Tn5-132 is also shown.

FIG. 3

Schematic representation of the two possible cointegrate structures (structures 1 and 2) between the integration plasmid pTH455 and a Tn5 transposon, and an agarose gel demonstrating both integration events as determined by PCR. Homologous recombination of pTH455 can occur at either of the two IS50 sequences, IS50R (structure 1) or IS50L (structure 2). The PCR primers (a, b, and c) are indicated by half arrows. The dashed line represents pExo DNA. Genomic DNA preparations of four integrants (I to IV) were used for PCR with primer sets a-b and b-c. Sample III carries pTH455 in IS50L, whereas the other three samples carry pTH455 in IS50R. Determination of pTH509 integration into both IS50 elements of Tn5-132 transposons is accomplished in a similar fashion with different primer sets. L, DNA ladder; pBS, pBluescript.

FIG. 4

Circular map of the pExo megaplasmid illustrating the relative locations of representative Tn5 derivative transposon insertions (Ω). The dashed line represents the region deleted in strain RmF909. The six regions (AA, AC, AD, AE, AF, and AG) which were flanked by the IS50-oriV-ΩSp-oriT cassettes and subsequently transferred to E. coli are indicated. The approximate sizes of the flanked regions are shown.

FIG. 5

Schematic representation of the four possible combinations of Ω5111::Tn5-oriT with Ω5142::Tn5-132::pTH509 (RmK255 to RmK258). RmK255 and RmK258 carry indirect oriT sites. Although RmK256 and RmK257 both carry direct (parallel) oriT sites, only in RmK257 do the oriT sites flank the chloramphenicol-resistant BAC vector (with the F origin of replication). A more detailed map of pTH509 is shown in Fig. 1. The dashed line represents pExo DNA. Cm^r, Nm^r, Ot^r, and Sp refer to chloramphenicol, neomycin, oxytetracycline, and spectinomycin resistance determinants, respectively.

FIG. 6

Southern blots demonstrate that the recovered plasmids in E. coli harbor S. meliloti DNA. Genomic DNA was prepared from wild-type strain Rm1021 and from deletion derivative RmF909. BamHI-restricted genomic DNA was hybridized with labeled pTH544 and pTH564 DNAs as indicated. The Rm1021 hybridization pattern closely resembles the probe pattern, showing that the plasmids carry pExo DNA. Moreover, DNA from RmF909, which lacks pExo DNA between Ω5085 and Ω5047 (Fig. 4), did not hybridize with either probe. BAC plasmid pTH544 carries DNA between Ω5159 and Ω5142 (Cm^r Sp^r Nm^s), and BAC plasmid pTH564 carries DNA between Ω5142 and Ω5102 (Cm^r Sp^r Nm^s).

FIG. 7

Subclones of the replicator region and a multiple alignment of the repB-repC intergenic region nucleotide sequences from large plasmids. Two subclones from the AA region, a 9.1-kb BamHI fragment (with the repABC genes) and a 4.4-kb BamHI/EcoRI fragment (with only the repC gene and repBC intergenic region), were transferred into A. tumefaciens (see text). An alignment of the repBC intergenic regions from several Agrobacterium and Rhizobium plasmid replicator regions is shown: the Ti plasmid (pTiB6S3) of A. tumefaciens B6S3 (51), the Ti plasmid (pTi-SAKURA) of A. tumefaciens MAFF301001 (50), the pExo megaplasmid of S. meliloti SU47 (this study), the Ri plasmid (pRiA4b) of A. rhizogenes (37), the symbiotic plasmid (p42d) of R. etli CFN42 (42), the symbiotic plasmid (pNGR234) of S. meliloti NGR234 (21), and the cryptic plasmid (pRL8JI) of R. leguminosarum (53). Hyphens indicate gaps introduced to give the best sequence alignments. Conserved nucleotide sequences are shaded; nucleotides identical for all seven sequences are indicated with asterisks.