Isolation of Auxotrophic Mutants of Methylophilus methylotrophus by Modified-Marker Exchange

Michael J. Bohanon, Cathryn A. Bastien, Roberta Yoshida, Richard S. Hanson
1988 Applied and Environmental Microbiology  
A method for stabilizing a transposon (TnS) has been developed which allows the isolation of stable auxotrophic mutants of Methylophilus methylotrophus AS1. Insertion of TnS into a cloned M. methylotrophus ASI DNA fragment encoding anthranilate synthase followed by transfer of the vector with the modified trpE gene to M. methylotrophus AS1 resulted in unstable auxotrophs among the recombinants. Deletion of ISSOR, which encodes transposase production from TnS, stabilized the transposon after
more » ... ransposon after mobilization to M. methylotrophus AS1. When trpE genes with the modified TnS inserts were mobilized into M. methylotrophus AS1, stable, kanamycin-resistant tryptophan auxotrophs were obtained by double-crossover homologous recombination with the chromosome.
doi:10.1128/aem.54.1.271-273.1988 fatcat:cpqvlxgwmnaejlbse6eee2pzua