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.

scholarly journals Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 244-253 ◽  
Author(s):  
Paola Sperandeo ◽  
Rachele Cescutti ◽  
Riccardo Villa ◽  
Cristiano Di Benedetto ◽  
Daniela Candia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
De Novo ◽  
Essential Genes ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
E Coli ◽  
Σ Factor

ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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.

scholarly journals Defects in The First Step of Lipoprotein Maturation Underlie The Synthetic Lethality of Escherichia coli Lacking The Inner Membrane Proteins YciB And DcrB

2021 ◽  
Author(s):  
Aaron Mychack ◽  
Anuradha Janakiraman
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Fluidity ◽  
Double Mutant ◽  
Synthetic Lethality ◽  
Cell Envelope ◽  
Initial Step ◽  
Inner Membrane ◽  
E Coli ◽  
Membrane Lipoprotein

Nearly a quarter of the Escherichia coli genome encodes for inner membrane proteins of which approximately a third have unassigned or poorly understood function. We had previously demonstrated that the synergy between the functional roles of the inner membrane-spanning YciB and the inner membrane lipoprotein DcrB, is essential in maintaining cell envelope integrity. In yciB dcrB cells, the abundant outer membrane lipoprotein, Lpp, mislocalizes to the inner membrane where it forms toxic linkages to peptidoglycan. Here, we report that the aberrant localization of Lpp in this double mutant is due to inefficient lipid modification at the first step in lipoprotein maturation. Both Cpx and Rcs signaling systems are upregulated in response to the envelope stress. The phosphatidylglycerol-pre-prolipoprotein diacylglyceryl transferase, Lgt, catalyzes the initial step in lipoprotein maturation. Our results suggest that the attenuation in Lgt-mediated transacylation in the double mutant is not a consequence of lowered phosphatidylglycerol levels. Instead, we posit that altered membrane fluidity, perhaps due to changes in lipid homeostasis, may lead to the impairment in Lgt function. Consistent with this idea, a dcrB null is not viable when grown at low temperatures, conditions which impact membrane fluidity. Like the yciB dcrB double mutant, dcrB null-mediated toxicity can be overcome in distinct ways - by increased expression of Lgt, deletion of lpp, or removal of Lpp-peptidoglycan linkages. The last of these events leads to elevated membrane vesiculation and lipid loss, which may, in turn, impact membrane homeostasis in the double mutant. Importance A distinguishing feature of Gram-negative bacteria is their double-membraned cell envelope which presents a formidable barrier against environmental stress. In E. coli, more than a quarter of the cellular proteins reside at the inner membrane but about a third of these proteins are functionally unassigned or their function is incompletely understood. Here, we show that the synthetic lethality underlying the inactivation of two inner membrane proteins, a small integral membrane protein YciB, and a lipoprotein, DcrB, results from the attenuation of the first step of lipoprotein maturation at the inner membrane. We propose that these two inner membrane proteins YciB and DcrB play a role in membrane homeostasis in E. coli and related bacteria.

Isolation and characterization of a bacteriophage infective for a UDP-galactose-4-epimeraseless mutant of Escherichia coli

1975 ◽  
Vol 21 (8) ◽  
pp. 1217-1223 ◽  
Author(s):  
Christopher J. Pazoles ◽  
Charles F. Kulpa Jr.
Keyword(s):  
Escherichia Coli ◽  
Cell Membrane ◽  
Receptor Site ◽  
Bacterial Strains ◽  
Adsorption Studies ◽  
E Coli ◽  
Isolation And Characterization

A DNA bacteriophage, designated CP13, was isolated against Escherichia coli J5, a UDP-galactose-4-epimeraseless mutant of E. coli 0111:B4. Bacteriophage CP13 appears to be specific for rough bacterial strains. Adsorption studies with E. coli J5 grown with galactose show that the bacteriophage will not adsorb when complete lipopolysaccharide is present in the cell membrane. This indicates that lipopolysaccharide may be directly or indirectly involved with the receptor site for bacteriophage CP13. The bacteriophage DNA has a G + C content of 52%.

scholarly journals Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli

2005 ◽  
Vol 187 (6) ◽  
pp. 1913-1922 ◽  
Author(s):  
Anindya S. Ghosh ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Fluorescent Dyes ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
E Coli ◽  
Physiological Processes ◽  
Side Walls

ABSTRACT In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.

scholarly journals Molecular Characterization of Carbapenemase-Producing Escherichia coli and Salmonella in children with diarrhea in rural Burkina Faso.

2020 ◽  
Author(s):  
Rene DEMBELE ◽  
Ali Konaté ◽  
Issiaka Soulama ◽  
Wendpoulomdé A. D. Kaboré ◽  
Assèta Kagambèga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Burkina Faso ◽  
Treatment Options ◽  
Diffusion Method ◽  
Bacterial Strains ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Encoding Genes

Abstract Background In recent years, Carbapenemase-producing Enterobacteriaceae (CPE) resistance to antibiotics has dramatically increased leading to limitations of their treatment options. In the present study, we investigated the occurrence of carbapenemase-producing Escherichia coli and Salmonella in rural Burkina Faso, using bacterial strains obtained from previous studies. Results Diarrheagenic Escherichia coli (DEC) strains was identified using 16-plex Polymerase Chain Reaction (PCR), whereas antibiotic susceptibility was realized using the disk diffusion method. Furthermore, multiplex PCR assays were used to characterize bla KPC, bla VIM and bla IMP genes in carbapenemase-producing E . coli and Salmonella . The study highlighted high resistance rates of the identified bacteria to common antibiotics. Likewise, two strains of E . coli were imipenem resistant with Carbapenemase-encoding genes. The genes detected were Klebsiella pneumoniae carbapenemase (KPC), Verona integrin-encoded metallo-β-lactamase (VIM) and Imipenemase (IMP-2) reaching a rate of 40% each. However, no Carbapenemase-encoding genes were detected in Salmonella isolates. Conclusions This study showed that for a real-time infection control and prompt application of antimicrobial chemotherapy, characterization of carbapenemase-producing Enterobacteriaceae in patients is crucial.

scholarly journals First Isolation and Molecular Characterization of blaCTX-M-121-Producing Escherichia coli O157:H7 From Cattle in Xinjiang, China

2021 ◽  
Vol 8 ◽  
Author(s):  
Zhanqiang Su ◽  
Panpan Tong ◽  
Ling Zhang ◽  
Mengmeng Zhang ◽  
Dong Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogen ◽  
Virulence Gene ◽  
Multidrug Resistant ◽  
Escherichia Coli O157 ◽  
Bacterial Strains ◽  
E Coli ◽  
Uremic Syndrome ◽  
Cattle Farms

The bovine Escherichia coli O157:H7 is a major foodborne pathogen causing severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Cattle are recognized major reservoir and source of E. coli O157:H7. We investigated the antibiotic resistance, molecular profiles, and intrinsic relationship between 21 isolates of E. coli O157:H7 from cattle farms and slaughtering houses in Xinjiang. Using pulsed-field gel electrophoresis (PFGE) molecular typing, two types of PFGE were revealed through cluster analysis, including clusters I and II, with 66 and 100% similarity of PFGE spectra between 21 isolates. We also detected that 18 isolates (86%) carried at least one virulence gene, 16 isolates (76%) carried the eae gene, and 7 (33%) carried the stx1 + stx2 + eae + hly + tccp genes. Eighteen isolates were susceptible to antibiotics. Three isolates were resistant to antibiotics, and two were multidrug resistant. One of the two multidrug-resistant isolates detectably carried the blaCTX−M−121 gene. This is the first finding of the blaCTX−M−121 gene detected in E. coli O157:H7 isolated from cattle in Xinjiang. The blaCTX−M−121 gene is transferable between the bacterial strains via plasmid transmission. The results indicated that E. coli O157:H7 may have undergone clonal propagation in cattle population and cross-regional transmission in Xinjiang, China.

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.

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