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.With the rapid increase in the number of completed microbial genome sequences, we are entering an era in which interests in the manipulation and functional characterization of whole genomes are flourishing. Methods and techniques involved in the manipulation of large regions of genomes will increasingly become valuable tools (for example, in the generation of mosaic organisms with various catabolic and biosynthetic capabilities). Here we describe a new procedure to clone large (Ͼ100-kb) defined regions from the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti.S. meliloti is a free-living gram-negative soil bacterium whose symbiotic interaction with alfalfa results in the formation of nitrogen-fixing root nodules. The genome of S. meliloti strain SU47 consists of three large replicons, the largest of which is 3,500 kb in size and appears to be similar to a conventional bacterial chromosome (4,28,36). The two other replicons are referred to as megaplasmids (47). One is 1,350 kb in size and, because it carries nodulation and nitrogen fixation genes required for symbiosis, is referred to as pSym (5,33,46); the other is 1,700 kb and is designated pExo since it carries two large gene clusters required for the synthesis of exopolysaccharides (alternate designations include pRmeSU47b and pSymb [4,13,22,30,33]).In previous work, we constructed a genetic map of the pExo megaplasmid which consists of sequential Tn5-derivative transposon insertions linked to each other in transduction (16). Strains carrying pExo megaplasmid deletions between defined insertions were isolated, and a phenotypic analysis of these strains allowed us to identify several loci involved in utilization of the carbon sources dulcitol, ␤-hydroxybutyrate, lactose, rhamnose, and protocatechuate (17). Other known g...

The complete sequence of the 1,683-kb pSymB megaplasmid from the N 2 -fixing endosymbiont Sinorhizobium meliloti

“…The origin of replication of pSymB is predicted to lie within the repA1B1C1 gene cluster, and subclones of this region are sufficient to allow its autonomous replication in Agrobacterium (23). There is a second repAB gene set (but lacking the repC gene) (repA3B3) lying 131 kb from the repA1B1C1 cluster.…”

Mining the Metabiome: Identifying Novel Natural Products from Microbial Communities

“…If the sequence of the target gene cluster is known a variety of recombination-based techniques ( e.g. transformation-assisted recombination (TAR) in the budding yeast Saccharomyces cerevisiae (Kim et al, 2010; Larionov et al, 1997), Red/ET recombination in E. coli (Bian etal., 2012; Fu et al, 2012; Muyrers et al, 2001), and oriT -directed capture (Chain et al, 2000; Kvitko et al, 2013)) can now used to directly clone intact gene clusters from genomic DNA. For metagenomic samples, which are significantly more complex than individual genomes and lack sequence data, metagenomic cosmid or fosmid libraries containing 30–40 kb inserts are first constructed and large gene clusters are assembled using TAR from overlapping metagenomic clones (Kim et al, 2010).…”

An Improved Method for oriT -Directed Cloning and Functionalization of Large Bacterial Genomic Regions

“…Several techniques have been developed to bridge this technical gap. The oriT-directed capture system developed by Chain et al has advantages for the specific cloning of large genomic regions compared with site-specific recombinase-catalyzed "pop-out" and shortflank recombineering-based strategies (2,(5)(6)(7)(8)(9)(10). The system is based on the principle that recombination will occur between two directly oriented RP4 origin of transfer (oriT) sites during conjugative transfer (11).…”

“…Transfer is terminated upon reaching the second oriT site, which is then recombined with the initiating oriT in the E. coli recipient, thus generating a circular mini-F-based plasmid carrying the target genome region. The system developed by Chain et al (5) was used to clone specific regions in excess of 200 kb and does not rely on the location of flanking restriction sites required by RecE-based recombineering strategies (2,5). The oriT-directed capture system does not generate deletions in the target chromosome during capture, because only one strand of the genome is mobilized.…”