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.

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 Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

2006 ◽  
Vol 189 (5) ◽  
pp. 1627-1632 ◽  
Author(s):  
Maria D. Bodero ◽  
M. Carolina Pilonieta ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Enterotoxigenic Escherichia Coli ◽  
Start Codon ◽  
Inner Membrane ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

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

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.

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.

scholarly journals Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Christian Lorenz ◽  
Thomas J. Dougherty ◽  
Stephen Lory
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

scholarly journals Disruption of lolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal for Escherichia coli and Prevents Release of Lipoproteins from the Inner Membrane

2002 ◽  
Vol 184 (5) ◽  
pp. 1417-1422 ◽  
Author(s):  
Shin-ichiro Narita ◽  
Kimie Tanaka ◽  
Shin-ichi Matsuyama ◽  
Hajime Tokuda
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Amino Acid Sequences ◽  
Atp Binding ◽  
Nonpermissive Temperature ◽  
Inner Membrane ◽  
E Coli ◽  
Atp Binding Cassette Transporter ◽  
Membrane Lipoproteins ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter for E. coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.

scholarly journals Characterization of the Effects ofn-butanol on the cell envelope ofE. coli

2016 ◽  
Author(s):  
Eugene Fletcher ◽  
Teuta Pilizota ◽  
Philip R. Davies ◽  
Alexander McVey ◽  
Chris E. French
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Protrusion ◽  
Bacterial Strains ◽  
External Concentration ◽  
Inner Membrane ◽  
E Coli ◽  
Rational Engineering

ABSTRACTBiofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects ofn-butanol on the cell envelope ofEscherichia coliwere investigated. Using enzyme and fluorescence-based assays, we observed that 1% v/v n-butanol resulted in release of lipopolysaccharides from the outer membrane ofE. coliand caused ‘leakiness’ in both outer and inner membranes. Higher concentrations ofn-butanol, within the range of 2% – 10% (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.

Colicin translocation across the Escherichia coli outer membrane

2012 ◽  
Vol 40 (6) ◽  
pp. 1475-1479 ◽  
Author(s):  
Nicholas G. Housden ◽  
Colin Kleanthous
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Cell Envelope ◽  
Protonmotive Force ◽  
Protein Protein Interactions ◽  
Inner Membrane ◽  
E Coli ◽  
Group A

We are investigating how protein bacteriocins import their toxic payload across the Gram-negative cell envelope, both as a means of understanding the translocation process itself and as a means of probing the organization of the cell envelope and the function of the protein machines within it. Our work focuses on the import mechanism of the group A endonuclease (DNase) colicin ColE9 into Escherichia coli, where we combine in vivo observations with structural, biochemical and biophysical approaches to dissect the molecular mechanism of colicin entry. ColE9 assembles a multiprotein ‘translocon’ complex at the E. coli outer membrane that triggers entry of the toxin across the outer membrane and the simultaneous jettisoning of its tightly bound immunity protein, Im9, in a step that is dependent on the protonmotive force. In the present paper, we focus on recent work where we have uncovered how ColE9 assembles its translocon complex, including isolation of the complex, and how this leads to subversion of a signal intrinsic to the Tol–Pal assembly within the periplasm and inner membrane. In this way, the externally located ColE9 is able to ‘connect’ to the inner membrane protonmotive force via a network of protein–protein interactions that spans the entirety of the E. coli cell envelope to drive dissociation of Im9 and initiate entry of the colicin into the cell.

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