Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K. pneumoniae mutant were constructed. The Gam-deficient Red recombinase showed a reduced capacity for gene replacement compared with that of the complete Red recombinase. The efficiency of gene replacement in the ΔrecB mutant was 6-8 times higher than the wild-type strain, and the minimum length for the homologous extensions was reduced to 100 bp. These results indicate that Gam does inhibit the RecBCD exonuclease in K. pneumoniae, but that this inhibition is not stringent. Furthermore, mutation of recB presents a convenient and efficient method to enhance the Red recombinase assisted gene replacement in K. pneumoniae.
Keywords: Gam; Gene replacement; Klebsiella pneumoniae; RecBCD; Red recombinase.
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