title: Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.
abstract: 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.
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.|Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People's Republic of China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Chen
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wei
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.
authors.forename: Pengfu
authors.initials: P
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.|School of Life Science and Technology, ShanghaiTech University, Shanghai, People's Republic of China.
authors.forename: Jiping
authors.initials: J
authors.lastname: Shi
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.
authors.forename: Biao
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Pudong, Shanghai, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Hao
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000037:antagonists & inhibitors*; D017353:Gene Deletion; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D043211:Exodeoxyribonuclease V
keywords: Gam; Gene replacement; Klebsiella pneumoniae; RecBCD; Red recombinase