scholarly journals Mutational Analysis of the TolA C-Terminal Domain of Escherichia coli and Genetic Evidence for an Interaction between TolA and TolB

2002 ◽  
Vol 184 (16) ◽  
pp. 4620-4625 ◽  
Author(s):  
Jean François Dubuisson ◽  
Anne Vianney ◽  
Jean Claude Lazzaroni
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mutational Analysis ◽  
Membrane Stability ◽  
Genetic Evidence ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Terminal Domain ◽  
Suppressor Mutants

ABSTRACT The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Randi L. Guest ◽  
Daniel Samé Guerra ◽  
Maria Wissler ◽  
Jacqueline Grimm ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Essential Protein ◽  
Ftsh Protease ◽  
Acyl Chains ◽  
Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

scholarly journals Yersinia pestis Is Viable with Endotoxin Composed of Only Lipid A

2005 ◽  
Vol 187 (18) ◽  
pp. 6599-6600 ◽  
Author(s):  
Li Tan ◽  
Creg Darby
Keyword(s):  
Escherichia Coli ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Membrane Component ◽  
Gram Negative ◽  
Serovar Typhimurium

ABSTRACT Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of Escherichia coli and Salmonella enterica serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the Enterobacteriaceae, Yersinia pestis, can survive without Kdo in its LPS.

scholarly journals Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli

2008 ◽  
Vol 190 (13) ◽  
pp. 4460-4469 ◽  
Author(s):  
Paola Sperandeo ◽  
Fion K. Lau ◽  
Andrea Carpentieri ◽  
Cristina De Castro ◽  
Antonio Molinaro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Gram Negative Bacteria ◽  
Membrane Structures ◽  
Cellular Compartment ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Assembly Pathway

ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

scholarly journals Expression of the Plasmodium falciparum Immunodominant Epitope (NANP)4 on the Surface of Salmonella enterica Using the Autotransporter MisL

2002 ◽  
Vol 70 (7) ◽  
pp. 3611-3620 ◽  
Author(s):  
Fernando Ruiz-Pérez ◽  
Rocío León-Kempis ◽  
Araceli Santiago-Machuca ◽  
Guadalupe Ortega-Pierres ◽  
Eileen Barry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Secretory Pathway ◽  
Cell Epitope ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Terminal Domain ◽  
Serovar Typhimurium

ABSTRACT Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by the sec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 of Salmonella enterica and classified as an autotransporter due to its high homology to Escherichia coli AIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) from Plasmodium falciparum on the surface of Salmonella enterica serovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL β domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind the Escherichia coli heat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically inducible nirB promoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP in P. falciparum sporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria.

scholarly journals A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Kerrie L. May ◽  
Kelly M. Lehman ◽  
Angela M. Mitchell ◽  
Marcin Grabowicz
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Outer Membrane ◽  
Stress Responses ◽  
Stress System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Essential Components ◽  
Trafficking Defects

ABSTRACTGram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses inEscherichia coli. Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM)en routeto the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM.IMPORTANCEThe outer membrane built by Gram-negative bacteria such asEscherichia coliforms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx helpE. colisurvive the assault of copper on a key outer membrane assembly pathway.

scholarly journals Primary structure of major outer-membrane protein I (ompF protein, porin) of Escherichia coli B/r

1982 ◽  
Vol 203 (1) ◽  
pp. 33-43 ◽  
Author(s):  
R Chen ◽  
C Krämer ◽  
W Schmidmayr ◽  
U Chen-Schmeisser ◽  
U Henning
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Outer Membrane ◽  
Primary Structure ◽  
Transmembrane Proteins ◽  
Low Molecular Weight ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Preliminary Communication ◽  
Escherichia Coli B

In the outer membrane of Gram-negative bacteria hydrophilic pores exist, allowing the diffusion of various low-molecular-weight solutes. These pores are formed by proteins, the porins. In a preliminary communication [Chen, Krämer, Schmidmayr & Henning (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5014-5017] we presented the primary structure of one of these porins, the 340-amino-acid-residue protein I (ompF protein) from Escherichia coli B/r. In the present paper we give the experimental evidence for this sequence. Two tryptophan positions, one valine position, two aspartic acid positions and nine out of 82 amide determinations have been corrected. To aid further studies on this class of transmembrane proteins, the isolation of most of the constituent peptides is documented.

scholarly journals An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Elayne M. Fivenson ◽  
Thomas G. Bernhardt
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Unknown Function ◽  
Barrier Function ◽  
Major Determinant ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Essential Protein

ABSTRACT Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share an intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adapter protein LapB (YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM (PbgA) participates in this regulatory pathway. Suppressors of YejM essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of ΔyejM mutants. Furthermore, the stability of LpxC was shown to be reduced in cells lacking YejM, and genetic and physical interactions between LapB and YejM were detected. Taken together, our results are consistent with a model in which YejM directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis. IMPORTANCE The outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this study, we identified an essential protein of unknown function as a key new factor in modulating LPS synthesis in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

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