Identification Of Genes Encoding Acyltransferases Of Lipid A In Pseudomonas Putida

Lipid A,which widely distributes on the outer leaflet of outer membranes in most Gram-negative bacteria,plays an important role in the survival and adaptation of bacterial cells.Pseudomonas putida is gram-negative bacteria which widely exists in the nature with application value in the restoration of the environmental pollution and synthesis of bioplastics polyhydroxyalkanoate.The secondary fatty acid chains of lipid A play a key role in the cell membrane surface hydrophobicity and antibiotic resistance.However,there was little study of the secondary fatty acid chains of lipid A in P.putida at present.This study made P.putida KT2442 as the research object,and found two genes encoding lipid A secondary acyltransferase through the sequence alignment.Moreover,through the deletion and overexpression of related genes,the specific function of two transferases in the synthesis of P.putida lipid A was confirmed.The main research results were described as follows:?1?Two proteins PP0063 and PP1735 of P.putida KT2442 have been identified by BLAST search with acyltransferase of Escherichia coli Lpx L,Pseudomonas aeruginosa PA0011,P.aeruginosa PA3242 and Acinetobacter baumannii LpxM,with corresponding genes PP₀063 and PP₁735,respectively.?2?Mutant strains KWZ001 and KWZ002 were constructed by deleting either genes PP₀063 and PP₁735 by homologous recombination.ESI/MS and TLC analysis of lipid A isolated from P.putida KT2442,KWZ001 and KWZ002 found that the lack of PP₀063 gene caused the lipid A of KWZ001 bacterium losing a hydroxy fatty acids chain,and loss of PP₁735 gene caused the lipid A of KWZ002 bacterium losing a fatty acids chain,which proved PP0063 and PP1735 as lipid A secondary acyltransferases.?3?Overexpressed strains with PP₀063 and PP₁735 in the mutant strains of KWZ001 and KWZ002 were constructed.ESI/MS and TLC analysis of lipid A isolated from expression strains KWZ001/pB-0063,KWZ001/pB-1735,KWZ002/pB-0063 and KWZ002/pB-1735 found that PP₀063 was unable to cover PP₁735 deleted strain,and PP₁735 cannot cover PP₀063 deleted strain,which showed the substrate specificity of PP0063 and PP1735.?4?In order to further determine the role of PP0063 and PP1735 in lipid A and substrate specificity,two genes lpxL and pagP which encode secondary acyltransferases with substrate specificity in E.coli MG1655 were heterologously expressed in mutant strains KWZ001 and KWZ002,respectively.ESI/MS and TLC analysis of lipid A isolated from expression strains KWZ001/pB-lpxL,KWZ001/pB-pagP,KWZ002/pB-lpxL and KWZ002/pB-pagP identified that PP0063 is responsible for adding 2-OH-C12:0 to C2 of lipid A,while PP1735 is responsible for adding C12:0 to C2' of lipid A.?5?Two knockout mutant strains KWZ001 and KWZ002 were studied in the different growth characteristics responsing to environmental change.It was confirmed that lack of secondary fatty acid chain did inhibit bacterial growth in the acid environment and low temperature environment.By RT-PCR we found that acidic conditions can activate the expression of EptA but low temperature conditions lower the activity of PagL.Further study showed that lack of secondary fatty acid chains of lipid A in P.putida affected the function ofthe cell membrane;improved the cell outer membrane permeability;reduced the cell aggregation ability;and to a certain extent,affected the bacteria for cationic antimicrobial peptides and antibiotic resistance.