Functional Analysis Of High-abundance Secreted Protein NJ7G＿0991 From Haloarchaeon Natrinema Sp.J7-2

NJ7G＿0991 is a secreted protein of halophilic archaea Natrinema sp.J7-2through Sec pathway,which has a very high abundance in the extracellular components.Bioinformatics analysis showed that it is a putative lipoprotein carrier protein with a Lol A-like structure domain.During the previous studies,nj7g＿0991deletion type I,whitch is partially destructed inside the gene nj7g＿0991,can lead to severe changes in the cell morphology and growth status.This suggests that the localization and assembly of S-layer and lipoproteins which are related to cell surface structure may be impacted.Thus,we further carried out an in-frame knock-out of nj7g＿0991 gene sequences within the Lol A domain-encoding fragment.The results showed that the deletion strain nj7g＿0991 deletion type II which obtained by in-frame deletion is unable to cause the changes of morphology and growth status.nj7g＿0990 is located in the downstream of nj7g＿0991 in the genome,sequence analysis showed that nj7g＿0990 and nj7g＿0991 share the same promoter,and NJ7G＿0990 has two copies of homologous domain refered to the universal stress protein(Usp),suggests that nj7g＿0990 may related to the induction and adaptation of Natrinema sp.J7-2 to the environmental pressures.Therefore,we further carried out the knock-out of nj7g＿0990,and constructed nj7g＿0991/0990 deletion type which is deletion in both the lol A domain encoding sequence within nj7g＿0991 and the two Usp domain encoding sequences within nj7g＿0990,beside the nj7g＿0990 deletion type which only delete the two Usp domain encoding sequence within nj7g＿0990.The results showed that there was no significant difference on cell morphology between the two deletion type strains and wild-type strain.Which means changes thatbroken out in nj7g＿0991 deletion type I may beyond the function of only two genes within nj7g＿0991 and nj7g＿0990.Further study on the multiple genes involved in the transport and localization of lipoprotein and their regulatory patterns in Natrinema sp.J7-2 is needed.When NJ7G＿0991 was recombinant and expressed in E.coli BL21(DE3),it was found that the recombinant NJ7G＿0991 protein may related to the assembly of E.coli outer membrane protein and lipopolysaccharide.The limited damage to the outer membrane of E.coli caused by NJ7G＿0991 can be used to further study its application potential in the construction of E.coli leakage expression system.In this paper,with the deletion and complementation experiments of nj7g＿0991and its adjacent gene,we confirmed that the deletion of nj7g＿0991 was not the cause of the specific characteristics of nj7g＿0991 deletion type I.The analysis of the protein composition in the extracellular supernatant of the recombinant strain of E.coli which expressed NJ7G＿0991 indicated that the effect of NJ7G＿0991 on the outer structure of E.coli may involve a variety of related lipoproteins.