Cloning And Sequence Analysis Of Phospholipase A₁ Gene From Serratia Marcescens CW-W-90-3 Strain

A genome library of Serratia marcescens CW-W-90-3 strain was established by "shot gun" method and screened for phospholipase activity on LB-egg yolk plate. One SOlObp EcoR I fragment containing phospholipase gene was isolated. Further sequence analysis and subcloning revealed a 963bp phlA. gene coding a 320aa phospholipase PHL with deduced molecular weight of 33KD. The PHL sequence shows high homology to the phospholipases AI from other bacteria. Another 756bp ORF was found downstream phi A. gene, coding for a 251aa polypeptide with molecular weight of 27KD. The protein designated as PHLS is highly homology to a phospholipase accessory protein, and is supposed to play important role in phospholipase AI expression. The phospholipase activity blotting of SDS-PAGE gel showed that Serratia marcescens CW-W-90-3 produce several phospholipases: p60 p33. p32 p31 p30 p29 p27 during growth. While only p60 and p27, which are supposed to be the mature phospholipases, existed when the S.m CW-W-90-3 enter the static phase. The p33, which is considered to be the precursor of p27 and coordinate to E. coli TGI expressed PHL, is expressed and excreted at lag phase. Then a splicing process is supposed occurred during the exponential phase resulting in production of mature phospholipase p27. The p32 p31 p30, p29 are believed to be intermediates of this splicing process. This phenomenon is not presented in E. coli. The p60 is expressed at middle to late exponential state and its activity is comparatively low.