Study On The Effect Of Dlt Operon On The Resistance Mechanism Of Polymyxin E In Bacillus Subtilis

The polymyxin is the last line of defense against multi-drug resistant,and has been widely used in the treatment of Gram-negative bacteria.In recent years,more and more studies showed that the cationic antimicrobial peptide polymyxin can also act on Gram-positive bacteria.With the application of polymyxin,a resistance mechanism against this cationic antimicrobial peptide had been found in Gram-positive bacteria.The dlt operon can increase the resistance to polymyxin E by modifying the positive charge on the cell surface by propionylation of the cell wall of Gram-positive bacteria.In this study,we focus on the interaction of polymyxin E and Bacillus subtilis,and study the role of the dlt operon in the resistance mechanism of polymyxin E.In this study,the dlt operon gene of Bacillus subtilis WB800 and the Escherichia coli-Bacillus subtilis shuttle plasmid pWBUC01 are digested by Xho I and Mlu I and then connected by ligase.The recombinant plasmid pWOP was transferred into Escherichia coli BW25113.Subsequently,the recombinant plasmid pWOP from E.coli was transferred into Bacillus subtilis SCK6 by Spizizen chemical transformation method,and Bacillus subtilis SCK6-2 with overexpressing dlt operon was successfully obtained.Compared with the MIC of polymyxin E to B.subtilis SCK6/pWBUC01?B.subtilis SCK6-1?and B.subtilis SCK6-2,we found that SCK6-2 is more tolerant to polymyxin E than SCK6-1,indicating that the overexpression of dlt operon contributes to the improvement of resistance to polymyxin E in B.subtilis.Compared with the growth curve and CFU of SCK6-1 and SCK6-2 under polymyxin E treatment,we found that the overexpression of dlt operon helps to improve the survival of B.subtilis treated with polymyxin E.In order to further investigate the reason that the overexpression of dlt operon can improve the resistance of B.subtilis to polymyxin E,we compared the OD_260nm and OD_280nm of the strain supernatant treated with polymyxin E.and the uptake of NPN.The results showed that the dlt operon attenuate the effect of polymyxin E on the permeability of B.subtilis.Subsequently,the cationic protein cytochrome c was used to examine the change in the net negative charge on the cell surface under overexpression of the dlt operon,and it was found that the overexpression of the dlt operon reduced the binding of B.subtilis to cytochrome c.This result indicates that overexpression of the dlt operon increases the positive charge on the surface of B.subtilis,reduces the net negative charge,and thus weakens the electrostatic binding of positively charged polymyxin E to cells.Bacillus polymyxa is the producing strain of polymyxin E and does not contain the dlt operon,and the expression of the dlt operon in B.polymyxa is expected to increase the resistance to polymyxin E.to increase the yield of polymyxin E in B.polymyxa.Therefore,this paper studied the expression and influence of the B.subtilis dlt operon in B.polymyxa.Firstly,the electroporation conditions of B.polymyxa ATCC842 and B.polymyxa C12 were optimized,and the optimal point transformation system was established.Next,a recombinant plasmid pWDA containing the dltA gene was constructed.Then,under the optimal system of electroporation,pWDA and pWOP were separately electrotransformed into Bacillus polymyxa ATCC842.It was found that pWDA could be successfully transformed,but the efficiency was relatively low;and pWOP could not be successfully transformed into strain ATCC842.The possible reason is that the electrotransformation efficiency of Bacillus polymyxa is relatively low.In addition,the dlt operon is much larger than dltA,which further increases the difficulty of pWOP electrotransformation.In the subsequent experiments,it is necessary to further improve the electrotransformation efficiency,and it is also possible to establish a more effective transformation method of Bacillus polymyxa.Therefore,the possibility of increasing the expression of the dlt operon to improve the resistance to polymyxin E and its mechanism provide a new idea for increasing the fermentation yield of polymyxin E.