Preliminary Study On The Function Of Cell Surface Proteins In Haloarchaeon Natrinema Sp.J7-2

The S layer is the surface structure of some bacteria and most archaea,which is composed of S layer protein subunits arranged in a crystalline repeat.The S layer protein is the first glycoprotein detected in prokaryotes and is still one of the most studied examples of glycosylated prokaryotes.The widespread presence of organisms with S layer,and the high abundance of S layer proteins reflects the adaptability of microorganisms to the natural environment and provides a competitive advantage for microorganisms under specific environmental and ecological conditions.In the previous study,through electron microscopy,the morphology of the haloarchaeon Natrinema sp.J7-2 cells and the tightness of the S layer structure were different under different salt concentrations.The S layer is the only component outside the Natrinema sp.J7-2 cell membrane.There's also a lot of lipoprotein on the cell membrane.The role of cell surface proteins in Natrinema sp.J7-2 were further studied in this paper.Based on the biological information analysis of Natrinema sp.J7-2 genomic data,five genes encoding S layer proteins were identified:nj7g₀389,nj7g₁525,nj7g₂776,nj7g₃860,and nj7g₃861.It was speculated that a variety of proteins together constituted S layer of Natrinema sp.J7-2.Mass spectrometric detection of Natrinema sp.J7-2 membrane fraction protein sample and extracellular secretory proteome was performed.Only NJ7G₀389 was not detected,and the other four proteins were detected.Under laboratory culture conditions,only NJ7G₁525 detected a distinct protein band by SDS-PAGE.Gene knockout is one of the most direct methods for studying protein function.Therefore,the nj7g₁525 gene was knocked out stepwise in the wild type and uracil-deficient strains by the "pop-in&pop-out" method to study the deletion of the nj7g₁525 gene and the S-layer defect on the impact of Natrinema sp.J7-2.The experimental results show that even if a portion of the nj7g₁525 gene is knocked out,no successful knockout strain can be obtained both in the wild type strain and the uracil-deficient strain.In this experiment,the knockout gene nj7g₁525 was found to cause cell death,which indirectly proved that the protein NJ7G₁525 plays an important role in the Natrinema sp.J7-2 as a key component of the S layer and is an indispensable part of its cell structure.The genomic analysis revealed that there are many related genes in the J7-2 strain that encode the lipoprotein-transporting Lol system,such as lolA,lolC,lolD,and lolE.The nj7g₀991 gene corresponds to lolA,and encodes a lipoprotein transporter in the J7-2 strain.It is speculated that NJ7G₀991 protein plays an important role in the lipoprotein transport and localization of the Natrinema sp.J7-2.Previous studies have found that nj7g₀991 defective type can normally grow,but the shape of the cells is changed compared to wild type strains.It is speculated that this mutation may have some influence on the plasma membrane and S layer.Since the maturation of S layer requires the magnesium ions,the growth conditions of the wild type J7-2 strain and nj7g₀991 defective type under different culture conditions of magnesium ion concentration were studied.It was found that lower Mg2+concentration?20 mM?would seriously affect cell growth.The wild type strain was more tolerant to EDTA and Triton X-100 than the nj7g₀991 defective type.Through EDTA mediated transformation efficiency tests of the two strains,it was found that the conversion rate of the wild type strain was significantly less than that of the nj7g₀991 defective strain.We speculated that the deletion of nj7g₀991 may affect the transport and localization of surface lipoproteins,thereby affecting the integrity of the membrane structure and making the exogenous gene fragments are more likely to enter cells,demonstrating that surface lipoproteins have a protective effect on cells.In addition,the chelation of magnesium ions by EDTA destroys the S layer,making it easier for foreign gene fragments to enter cells.The above results show that the cell surface proteins have a certain protective effect.