Identification Of High-abundance Secreted Proteins And Functional Analysis Of An Extracellular Protein NJ7G₀991 From Haloarchaeon Natrinema Sp.J7-2

The protein secretion is one of the most important biological process.There are two pathways that are widely used for protein secretion: Sec pathway and Tat pathway.Both Sec and Tat pathways exist in haloarchaea.The identification of secreted proteins of haloarchaea can not only provide useful information about the type,function and localization of them,but also be helpful to understand the adaptation mechanism of haloarchaea to high salt environments.In this study,we performed proteomic analysis of secreted proteins of the haloarchaeon Natrinema sp.J7-2.In addition,a highabundance secreted protein was subjected to functional analysis.Firstly,the culture supernatant and membrane fraction of strain J7-2 were subjected to RPLC-ESI-MS/MS analysis.Analysis of the data revealed that there are more Tat signal peptide-bearing secreted proteins,which function mainly as solutebinding proteins and redox proteins anchored to cytoplasmic membrane via lipobox.Sec signal peptide-bearing secreted proteins are mainly involved in the biogenesis of cell envelope and surface appendices.Meanwhile,many transmembrane proteins also contain Sec signal peptides,implying that the Sec pathway is also important for translocation and localization of membrane proteins.Notably,most secreted proteins were found in the membrane fraction.The anchoring of secreted proteins to the cell surface seems to be an adaptation strategy employed by haloarchaea to deal with the harsh extracellular environment.Secondly,three high-abundance extracellular proteins of strain J7-2 were identified as Sec substrates NJ7G?1525 and NJ7G?0991 and Tat substrate NJ7G?0566.The cell surface glycoprotein NJ7G?1525 belongs to the halo?surf?glyco family(TIGR04216),of which the members are haloarchaeal S-layer proteins.The presence of the high-abundance NJ7G?0566 with a halocyanin domain in the membrane fractions emphasizes the role of halocyanins in energy conversion of haloarchaea.Considering that Natrinema sp.J7-2 produces many putative lipoproteins,NJ7G?0991with a Lol A-like domain is likely to be involved in lipoproteins translocation and localization.Thirdly,we performed functional analysis of NJ7G?0991.The gene nj7g?0991 of strain J7-2 was knocked out by homologous recombination method.The loss of NJ7G?0991 with a Lol A-like domain not only led to a change in cell morphology of the haloarchaeon,but also decreased its tolerance to external stimuli and weakened its motility.The results showed that NJ7G?0991 played an important role in stabilizing cell envelope of Natrinema sp.J7-2.Finally,we found that the production of recominant NJ7G?0991 in Escherichia coli resulted in the damage of the outer membrane of E.coli.Most likely,the lol system of E.coli was disturbed by recombinant NJ7G?0991,leading to mislocalization of outer membanre lipoproteins of E.coli.The fucntional analysis of NJ7G?0991 may provide new clue for better understanding lipoprotein translocation and localization in haloarchaea.