A general method for site-directed mutagenesis in prokaryotes

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Abstract
A general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments, applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti [associated with Medicago sativa] is reported. Cloned R. meliloti restriction fragments in Escherichia coli were mutagenized with transposon Tn5 and the wild-type parental DNA sequences were replaced with the mutant DNA sequences in the R. meliloti genome. An R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. This method is used to construct a physical genetic map of a subset of the R. meliloti nif genes.