Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ

2020 ◽  
Vol 15 (3) ◽  
pp. 031003
Author(s):  
Johannes Kassmannhuber ◽  
Sergio Mauri ◽  
Mascha Rauscher ◽  
Nadja Brait ◽  
Lea Schöner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Inner Membrane ◽  
Membrane Bound

scholarly journals Freezing from the inside. Ice nucleation in <i>Escherichia coli</i> and <i>Escherichia coli</i> ghosts by inner membrane bound ice nucleation protein InaZ

2019 ◽  
Author(s):  
Johannes Kassmannhuber ◽  
Sergio Mauri ◽  
Mascha Rauscher ◽  
Nadja Brait ◽  
Lea Schöner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Pseudomonas Syringae ◽  
Survival Rates ◽  
Ice Nucleation ◽  
Free Access ◽  
Ice Nucleation Protein ◽  
Inner Membrane ◽  
E Coli ◽  
Subzero Temperatures

Abstract. An N-terminal truncated form of the ice nucleation protein (INP) of Pseudomonas syringae lacking the transport sequence for the localization of InaZ in the outer membrane was fused to N- and C- terminal inner membrane (IM) anchors and expressed in Escherichia coli C41. The ice nucleation (IN) activity of the corresponding living recombinant E. coli catalyzing heterogeneous ice formation of super-cooled water at high subzero temperatures was tested by droplet freezing assay. Median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at −20.1 °C and with inner membrane anchored INPs at T50 value between −7 °C and −9 °C demonstrating that IM anchored INPs facing the luminal IM site are able to induce IN from the inside of the bacterium almost similar to bacterial INPs located at the outer membrane. Bacterial Ghosts (BGs) derived from the different constructs showed first droplet freezing values between −6 °C and −8 °C whereas C41 BGs alone without carrying IM anchored INPs exhibit a T50 of −18.9 °C. The more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of IM anchored INP constructs to ultrapure water filling the inner space of the BGs. The cell killing rate of -NINP carrying E. coli at subzero temperatures is higher when compared to survival rates of the parental C41 strain.

Expression of Carboxymethylcellulase on the Surface of Escherichia Coli Using Pseudomonas Syringae Ice Nucleation Protein

1998 ◽  
Vol 22 (5) ◽  
pp. 348-354 ◽  
Author(s):  
Heung-Chae Jung ◽  
Joon-Hyun Park ◽  
Seung-Hwan Park ◽  
Jean-Michel Lebeault ◽  
Jae-Gu Pan
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Ice Nucleation Protein

scholarly journals Cell Surface Display of Human Immunodeficiency Virus Type 1 gp120 on Escherichia coli by Using Ice Nucleation Protein

1999 ◽  
Vol 6 (4) ◽  
pp. 499-503 ◽  
Author(s):  
Young-Don Kwak ◽  
Seung-Ku Yoo ◽  
Eui-Joong Kim
Keyword(s):  
Escherichia Coli ◽  
Human Immunodeficiency Virus ◽  
Surface Display ◽  
Cell Surface Display ◽  
Viral Proteins ◽  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface ofEscherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.

Membrane assembly: movement of phosphatidylserine between the cytoplasmic and outer membranes of Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 1033-1041
Author(s):  
K E Langley ◽  
E Hawrot ◽  
E P Kennedy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sucrose Gradient ◽  
Intracellular Localization ◽  
Temperature Sensitive ◽  
Inner Membrane ◽  
E Coli ◽  
Outer Membranes ◽  
Membrane Bound ◽  
Gradient Fractionation

Phosphatidylserine, normally a trace phospholipid in Escherichia coli, accumulates at high levels in temperature-sensitive phosphatidylserine decarboxylase mutants at nonpermissive temperatures. The intracellular localization of this phospholipid has now been determined. All of the accumulated phosphatidylserine is membrane bound and is distributed about equally between the inner and outer membrane fractions of E. coli as determined by isopycnic sucrose gradient fractionation. Phosphatidylserine is therefore effectively translocated from the inner to the outer membrane. Furthermore, this movement is bidirectional. Outer membrane phosphatidylserine can return to the inner membrane, as shown by the complete conversion of accumulated radioactive phosphatidylserine to phosphatidylethanolamine by inner membrane phosphatidylserine decarboxylase during chase periods. Pulse-chase experiments indicated the newly made phosphatidylserine appears first in the inner membrane and then equilibrates between the inner and outer membranes with a half-time of 12 to 13 min.

Expression of xylanase on Escherichia coli using a truncated ice nucleation protein of Erwinia ananas (InaA)

2019 ◽  
Vol 78 ◽  
pp. 25-32 ◽  
Author(s):  
Mei Yuin Joanne Wee ◽  
Abdul Munir Abd. Murad ◽  
Farah Diba Abu Bakar ◽  
Kheng Oon Low ◽  
Rosli Md Illias
Keyword(s):  
Escherichia Coli ◽  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Erwinia Ananas

scholarly journals Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

2009 ◽  
Vol 76 (2) ◽  
pp. 434-440 ◽  
Author(s):  
Chao Yang ◽  
Roland Freudl ◽  
Chuanling Qiao ◽  
Ashok Mulchandani
Keyword(s):  
Escherichia Coli ◽  
Methyl Parathion ◽  
Ice Nucleation ◽  
Organophosphorus Hydrolase ◽  
Display System ◽  
Ice Nucleation Protein ◽  
Methyl Parathion Hydrolase ◽  
E Coli ◽  
Tat Pathway ◽  
Parathion Hydrolase

ABSTRACT A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.

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