Simultaneous overabundance and scarcity of inorganic phosphate(Pi)is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrop...Simultaneous overabundance and scarcity of inorganic phosphate(Pi)is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication,but also recover Pi for agricultural reuse.Here,a cell-surface expressed high-affinity phosphate binding protein(PstS)system was developed,and its Pi capture and release potential was evaluated.E.coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein(INP)as an anchoring motif.Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis(SDS-PAGE),western blot,and outer membrane separation analyses.Cell surface characterization was investigated through acid-base titration,X-ray photoelectron spectroscopy(XPS),and Fourier transform infrared spectroscopy(FTIR).These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups(i.e.,pKa values).Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature,pH,and ionic strength on phosphate capture and release.The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells.Higher temperatures up to 40℃,basic pH conditions(pH=10.5),and higher ionic strength up to 1.0 mol/L KCl promoted 20%-50%higher phosphate release.展开更多
Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of ...Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of animals and plants, Pst DC3000 exploits the conserved type III secretion system (TTSS) to deliver multiple virulence effectors directly into the host cells. Type III effectors (T3Es) collectively participate in causing disease, by mechanisms that are not well clarity. Elucidating the virulence function of individual effector is fundamental for understanding bacterial infection of plants. Here, we focused on studying one of these effectors, HopAA1-1, and analyzed its potential function and subcellular localization in N. benthamiana. Using an Agrobacterium-mediated transient expression system, we found that HopAA1-1 can trigger domain-dependent cell death in N. benthamiana. The observation using confocal microscopy showed that the YFP-tagged HopAA1-1 localizes to diverse cellular components containing nucleus, cytoplasm and cell membrane, which was demonstrated through immunoblot analysis of membrane fractionation and nuclear separation. Enforced HopAA1-1 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that HopAA1-1-induced cell death in N. benthamiana is suppressed in the nucleus but enhanced in the cytoplasm. Our research is lay a foundation for revealed the molecular pathogenesis of Pseudomonas syringae pv. tomato.展开更多
基金supported by CAREER award 1554511 from the National Science Foundation(NSF)。
文摘Simultaneous overabundance and scarcity of inorganic phosphate(Pi)is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication,but also recover Pi for agricultural reuse.Here,a cell-surface expressed high-affinity phosphate binding protein(PstS)system was developed,and its Pi capture and release potential was evaluated.E.coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein(INP)as an anchoring motif.Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis(SDS-PAGE),western blot,and outer membrane separation analyses.Cell surface characterization was investigated through acid-base titration,X-ray photoelectron spectroscopy(XPS),and Fourier transform infrared spectroscopy(FTIR).These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups(i.e.,pKa values).Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature,pH,and ionic strength on phosphate capture and release.The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells.Higher temperatures up to 40℃,basic pH conditions(pH=10.5),and higher ionic strength up to 1.0 mol/L KCl promoted 20%-50%higher phosphate release.
文摘Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of animals and plants, Pst DC3000 exploits the conserved type III secretion system (TTSS) to deliver multiple virulence effectors directly into the host cells. Type III effectors (T3Es) collectively participate in causing disease, by mechanisms that are not well clarity. Elucidating the virulence function of individual effector is fundamental for understanding bacterial infection of plants. Here, we focused on studying one of these effectors, HopAA1-1, and analyzed its potential function and subcellular localization in N. benthamiana. Using an Agrobacterium-mediated transient expression system, we found that HopAA1-1 can trigger domain-dependent cell death in N. benthamiana. The observation using confocal microscopy showed that the YFP-tagged HopAA1-1 localizes to diverse cellular components containing nucleus, cytoplasm and cell membrane, which was demonstrated through immunoblot analysis of membrane fractionation and nuclear separation. Enforced HopAA1-1 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that HopAA1-1-induced cell death in N. benthamiana is suppressed in the nucleus but enhanced in the cytoplasm. Our research is lay a foundation for revealed the molecular pathogenesis of Pseudomonas syringae pv. tomato.
