Study On The Structure Diversity And Gene Functions Of Lipid A In Pseudomonas Putida KT2442

The important industrial bacterial Pseudomonas putida KT2442 is Garm-negative bacterial with large amount of lipopolysaccharide（LPS） molecules distributed around the cell surface, but the detailed structure of LPS has not been reported yet. This study identified the structure and composition of phospholipid and lipid A, as well as the gene function related to lipid A by molecular biology and chemistry biology methods. The main results were described as follows:（1） The phospholipid and lipid A were successfully isolated from P. putida KT2442 and its structure was analyzed by TLC, HPLC, ESI/MS, and ESI/MS/MS. The major phospholipid in KT2442 is composed of phosphatidylethanolamine（79.9%）,phosphatidylglycerol（12.7%） and cardiolipin（7.4%） with C16:1 and/or C18:1 acyl chains.At least six different lipid A structures were identified in P. putida whose fatty acid chains are made up of C₁₀ and C₁₂.（2） Both PP₀737 and PP₃154 can encode lipid A 3-O-deacylase which can remove the fatty acid in 3-position, thus these two genes were renamed pag L1 and pag L2,respectively. At first, we expressed the two genes in E. coli W3110 and HW001 to identify the gene function. Then we knockout pag L1 and pag L2 in KT2442 to further prove both of the two genes can encode the lipid A 3-O-deacylase. Finally, we expressed these two genes in KT2442 and knockout strains to verify our above result.（3） The influence of different environment on the transcription level of pagL1 and pagL2 and level of deacylation have been systemically studied. The transcription level of pag L1 was decreased by adding high concentration Zn2+ and Mg2+; the transcription level of pag L2 was increased by adding high concentration Zn2+, low concentration Mg2+ and 0.01% xylene. Besides, the temperature and Zn2+ mainly effected the enzyme activity of Pag L1 and Pag L2.The level of deacylation increased with the temperature and concentration of xylene, while decreased with the concentration of Mg2+ and Zn2+.（4） The influence of Pho P-Pho Q and ColR-ColS on the level of deacylation and transcription level of pag L1 and pag L2 have been systemically studied. Pho P-Pho Q can enhance the deacylation of lipid A when KT2442 were cultured in N-mineral medium with 1 mmol/L Mg2+ or 10 μmol/L Mg2+; Col R-Col S can slightly reduce the deacylation of lipid A when cultured in LB and N-mineral medium with 1 mmol/L Mg2+, and enhance the deacylation in LB medium with 0.01% xylene. Besides, the regulation of Col R-Col S was not stable when cultured in N-mineral medium with 10 μmol/L Mg2+.（5） Both PP₂423 and PP₄570 can encode lipid A dioxygenase which can add hydroxyl to the secondary fatty acid in 2-position. Thus, these two genes were renamed lpx O1 and lpx O2, respectively. PP₁735 can encode lipid A acyltransferase which can add C₁₂ fatty acid in 2’-position. Thus, PP₁735 was renamed lpx L2. Moreover, PP₀024 can encode lipid A phosphoethanolamine transferase which can add pet N in 1’ or 4’-position, for which reason PP₀024 was renamed eptA.