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Here we show that the frequency of mutant isolation by two-step allele exchange can be improved by increasing the length of homologous DNA and the opportunity for recombination, obviating the need for counterselection markers. These principles are demonstrated in Clostridium difficile and Streptococcus suis but are likely to be generally applicable.

Improved Bacterial Mutagenesis by High-Frequency Allele Exchange, Demonstrated in Clostridium difficile and Streptococcus suis