Development of a broad-host-range sacB-based vector for unmarked allelic exchange

Abstract

Background: Although genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models.

Results: Here I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433.

Conclusion: The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired.

Figure 1

Strategy for pCM433-based "in-out" allelic exchange. Scheme depicted for the example of introducing a mutant allele with a point mutation (orf^mut) in place of the wt allele (orf^wt). PCR amplification of orf^mut with primers complementary to the boundaries of the coding sequence, followed by cloning of the resulting fragment into pCM433, results in the desired orf^mut donor plasmid. This donor can then be conjugated into the orf^wt recipient strain, for which selection of one of the three encoded antibiotic resistances (Ap, Cm, Tc) will result in strains that have experienced recombination at the desired allele, and incorporation of the donor plasmid into the chromosomal locus. Although the recombination is drawn here to have occurred to the right of the point mutation, resulting in the orf^wt upstream, recombination to the left of the point mutation would situate orf^mut upstream. Selection for sucrose resistance, and screening for antibiotic sensitivity results in clean exchange of the orf^mut allele or reversion to orf^wt. Finally, isolates bearing clean, single copies of the locus can be screened to identify a strain bearing the desired allele.

Figure 2

Broad-host-range allelic exchange vector pCM433. Plasmid map of pCM433 [GenBank:EU118176] indicating the available restriction sites in the multiple-cloning site, as well as key plasmid features: bla (encodes betalactamase; Ap^R), oriT (IncP origin of conjugal transfer), tet (encodes Tc efflux pump), cat (encodes Cm actetyltransferase), sacB (levansucrase; confers sucrose sensitivity), and ColE1 ori (high copy E. coli origin of replication). Plasmid map was generated using Clone Manager 7 (Sci Ed Central).