Phenotypic expression of PCR-Generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also k nown as strain BD413) by natural transformation. The technique does not req uire cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homol ogous recombination. Previously such procedures for random mutagenesis coul d be applied only to Acinetobacter genes affording the selection of mutant phenotypes, Here we describe the construction of a vector and recipient tha t allow for mutagenesis, recovery, and expression of heterologous genes tha t may lack a positive selection. The plasmid carries an Acinetobacter chrom osomal segment interrupted by a multiple cloning site next to a kanamycin r esistance marker. The insertion of heterologous DNA into the multiple cloni ng site prepares the insert as a target for PCR mutagenesis, PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity bet ween the flanking regions and corresponding chromosomal segments in an engi neered Acinetobacter recipient allows homologous recombination of the PCR-a mplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the m utagenized insert can be selected by demanding growth of recombinants in th e presence of kanamycin. The effectiveness of the technique was demonstrate d with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C . Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C, The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.