Phosphatidyl Choline Affect On Beta-lactamase Secretion Of Pseudomonas Sec-dependent

Sec secretion system is also known as sec pathway or the general secretory pathway. It is a kind of secreted machine to secret protein, located in the inner membrance of gram-negative bacterias. Proteins Secreted by the sec system with a signal peptide sequence which can be specificlly recognized by receptor, and combined with it so that guide the protein transmembrane transport. Sec transporter enzyme is a multi-protein complex component, the core of transporter enzyme is composed with SecYEG and SecA.Integral membrane protein SecD, SecF and YajC form a complex subunit, which can be connected to the SecYEG and stable the membrane-bound form of the protein secA.Phosphatidylcholine (PC) is a critical structural component of cell membrane.It widely exists in animals, plants, humans and other eukaryotes, and a small number of prokaryotes also contain the PC. Pseudomonas syringae pv.Syringae1336and Pseudomonas sp.593can synthesis phosphatidyl choline by-Pcs-dependent pathway which depends on choline.Pseudomonas syringae pv.Syringae1336and Pseudomonas sp.593can secret beta-lactamase by sec system in inner membrane.This experiment will compare the secretion of beta-lactamase from Pseudomonas.sp593with Pseudomonas sp593pcs mutant and compare Pseudomonas syringae pv.Syringae1336with1336pcs mutant.Assaying the beta-lactamase activity, Western-blot and RT-PCR. So that we could analysis how PC impacts sec system. I will do this experiment as follows.(1) Testing the resistance of the Pseudomonas syringae pv.Syringae1336and Pseudomonas sp593wild type and pcs mutant to ampicillin. A preliminary inspection made by filter paper plate method tests the resistance of above four kinds of bacterias to Amp.The results show that the resistance of Pseudomonas syringae pv.Syringae1336and its pcs mutant,593and its pcs mutant to Amp are different. Then measure the half lethal dose(IC50) of Amp to above four kinds of bacteria.(2) Assaying the activity of beta-lactamase. First extract the Periplasmic proteins and cytoplasmic proteins from Pseudomonas syringae pv.Syringae1336and its pcs mutant, Pseudomonas sp593and its pcs mutant, use nitrocefin as the substrate, beta-lactamase activity is measured by spect-rophotometry. We find that the the beta-lactamase activity significantly different between1336and593wild-type and their mutants。(3) Gene cloning and expression for beta-lactamase that is secreted by sec system of Pseudomonas syringae pv. Syringae1336and Pseudomonas sp.593. we get six genesthat may encode beta-lactamase from Pseudomonas syringae pv. SyringaeB728a by searching NCBI.The primers were designed according to this. The total DNA of Pseudomonas syringae pv. Syringae1336and Pseudomonas sp.593as a template for PCR. only AmpC, psyr2741and psyr3855were cloned.The PCR product was sequenced and signal peptide prediction by SignalP,we can find the1-21amino acid residues of AmpC may as signal peptide. Comparative analysis of the characteristics of the signal peptide, we find that the signal peptide can be specific recognition by Sec secretion system. That is to say the precursor protein with the signal peptide may be secreted by the Sec system.While the same specific signal peptide sequence can not be find in Psyr2741, Psyr3855.(4) Preparation Polyclonal antibody and Western-bloting. construction of the expression vector of AmpC, psyr2741, psyr3855gene and transformed into E. coli BL21(DE3),and make their expression then purified corresponding proteins. preparation Polyclonal antibody by injected proteins into subcutaneous of rabbits. Detected AmpC,Psyr2741and Psyr3855in cytoplasm and periplasm of1336and593wild-type and their mutants by Western-bloting. The results showed that there are more AmpC in periplasm of mutant strains than wild-type,but there are no significant difference in the cytoplasm of wild-type strains and their mutants. While the protein Psyr2741, Psyr3855were detected only a small amount in cytoplasm and failed to be detected in periplasm of wild-type strains and their mutants.(5) Refolding AmpC, Psyr2741, Psyr3855inclusion body and activity assay. Collecting the three purified proteins inclusion body refolding the inclusion body in refolding solution by gradient dialysis about36hours and then the supernatant was collected.the result showed that Refolded protein only AmpC has β-lactamase activity that can hydrolysis Nitrocefin.(6) The expression of AmpC, psyr2741, psyr3855was detected by RT-PCR. Extract the total RNA from1336and its pcs mutant,593and its pcs mutant, the cDNA is synthesized by reverse transcription, the16SRNA uses as standards, by doing PCR to determine whether the transcription of beta-lactamase is influenced by the lack of pcs gene. Three proteins found in these four strains were expressed, and its expression level in wild-type and mutant strains were no significant differences.To make a conclusion, the first one is only AmpC gene encoding β-lactamase in the three genes from1336and593and AmpC can be secreted by Sec.And the ability of the mutants to anti-Amp is higher than the wild type mainly because of AmpC is secreted periplasmic space of mutant more than wild type. And the second one is AmpC is secreted different in wild type and mutants because of PC,That is to say PC can effect the secretion of beta-lactamase by the Sec system.