An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis.
A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.
Ried
J L
JL
Department of Botany, University of Maryland, College Park 20742.
Collmer
A
A
D004885:Erwinia / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005819:Genetic Markers; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007613:Kanamycin Resistance; D009154:Mutation; D010957:Plasmids
D005819:Genetic Markers