Use of the Caulobacter crescentus genome sequence to develop a method for systematic genetic mapping

The functional analysis of sequenced genomes will be facilitated by the development of tools for the rapid mapping of mutations. We have developed a systematic approach to genetic mapping in Caulobacter crescentus that is based on bacteriophage-mediated transduction of strategically placed antibiotic resistance markers. The genomic DNA sequence was used to identify sites distributed evenly around the chromosome at which plasmids could be nondisruptively integrated. DNA fragments from these sites were amplified by PCR and cloned into a kanamycin-resistant (Kan r ) suicide vector. Delivery of these plasmids into C. crescentus resulted in integration via homologous recombination. A set of 41 strains containing Kan r markers at 100-kb intervals was thereby generated. These strains serve as donors for generalized transduction using bacteriophage Cr30, which can transduce at least 120 kb of DNA. Transductants are selected with kanamycin and screened for loss of the mutant phenotype to assess linkage between the marker and the site of the mutation. The dependence of cotransduction frequency on sequence distance was evaluated using several markers and mutant strains. With these data as a standard, previously unmapped mutations were readily localized to DNA sequence intervals equivalent to less than 1% of the genome. Candidate genes within the interval were then examined further by subcloning and complementation analysis. Mutations resulting in sensitivity to ampicillin, in nutritional auxotrophies, or temperature-sensitive growth were mapped. This approach to genetic mapping should be applicable to other bacteria with sequenced genomes for which generalized transducing phage are available. Complete genomic DNA sequences have generated fascinating insights into the biology and evolutionary history of many bacterial species. Genome sequences have also enabled powerful new technologies such as microarrays to be applied to understanding the activities of microorganisms (25). Sometimes overlooked is the fact that genomic sequence information can be very productively combined with traditional methods of genetic analysis. We have used the recently completed Caulobacter crescentus genome sequence (17) as a guide to produce a set of genetic mapping strains suitable as donors for generalized transduction. Using these mapping strains, mutations with a variety of phenotypes can be quickly and reliably localized to a small fraction of the genome, identifying candidate genes for further investigation. This technique will significantly speed up genetic analysis of C. crescentus and is likely to be applicable to other bacterial species as well. C. crescentus is a gram-negative aquatic bacterium that serves as a model system for studying the bacterial cell cycle and development (19). Caulobacter exhibits two distinct appendaged cell types—a motile swarmer cell with a single polar flagellum and a nonmotile cell displaying an adhesive stalk. Swarmer cells can be isolated by density centrifugation to produce synchronized cultures that move through the cell cycle in unison. Swarmer cells differentiate into stalked cells by ejecting