Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein.
The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand. stwcj@mucc.mahidol.ac.th
Wipa
W
Chungjatupornchai
Sirirat
S
Fa-aroonsawat
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D046940:Synechococcus / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D049452:Green Fluorescent Proteins