Preliminary Study Of Gene Knockout In K. Pneumoniae

Klebsiella pneumoniae is a Gram-negative bacteria which containing a large number of capsular polysaccharide(CPS).There are mainly two serotypes of Klebsiella pneumoniae:K1 serotype and K2 serotype, based on the characteristics of CPS. The K1 serotype strains essentially contain magA gene, and almost all of Klebsiella pneumoniae strains contain four conservative gene:orfX((wzi), wza, wzb and wzc, which are relative to the transfer and surface assembly of capsular polysaccharide.Red recombinantion system is an efficient system for gene knockoutting of E. coli K12 strains, it aiso has been confirmed in some other bacteria strains. The method of gene knockout in Klebsiella pneumoniae is still based on the suicide plasmid of oriR6K replicon. There is no report about the application of Red recombination system in Klebsiella pneumoniae.In this study we have amplified the magA, wzi and wzc gene according to the Klebsiella pneumoniae NTUH-K2044 strain’s cps gene (accession number AB 198423.1), and found wzi gene is a highly conserved homologous gene. It can be a good target in the study of gene knockout in Klebsiella pneumoniae. We have constructed the plasmid pKD46-Tc and pCP20-Tc which containing tetracycline resistance marker, and established a Red recombination system in Klebsiella pneumoniae. We constructed a gene-targetting fragment called A wzi::FT which contained 500-700bp homologous segments and a fragment called A wzi::FK which contained 250bp homologous segments. A wzi::FT had been transformed into Klebsiella pneumoniae without Red recombination system, and A wzi::FK had been transformed into Klebsiella pneumoniae/pKD46-Tc. compared the result of them, we found that the former had no recombinant, but the latter resulted six recombinants, we could estimate that the capsule gene wzi maybe had been knockout by using the Red recombination system in Klebsiella pneumoniae.