From proteome to genome for functional characterization of pH-dependent outer membrane proteins in Escherichia coli

Proteomic technology is very powerful in identification of differentially expressed proteins. However, how to identify key proteins and distinguish them from others has been a question to be solved in functional proteomics. Utilizing 2-D gel based proteomic approach, we identified 11 differentially expressed outer membrane (OM) proteins involved in E. coli's response to pH change. The protein expression changes were validated by Western blotting. The function and roles of the differentially expressed proteins were further characterized using genetically modified strains with gene deletion of these altered OM proteins and gene complementation or overexpression approach. Among the differentially expressed proteins identified, OstA, TolC, OmpT, OmpP OmpC, Trak, OmpX, Dps, LamB, Tsx, FadL, OmpW, and OmpF were characterized as pH-related OM proteins. Out of these OM proteins, TolC, OmpC, OmpX, and LamB may play critical roles in pH-regulation in E. coli. Using death-rescuing assay developed in house, we found that OmpC, LamB, FadL, OmpX, OmpW, and OmpF, LamB, FadL, and OmpW functioned in a TolC-independent pathway, whereas OmpF, Tsx and OmpC, OmpX, and Tsx might share the same pathway with TolC at the extreme acid or base condition. The information obtained from this study provides novel insight into mechanisms of pH response in E. coli. Our results also demonstrate the importance and efficiency of functional characterization of differentially expressed proteins at different molecular levels in identification of key target proteins and pathways involved in E. coli' s response to pH change.