Crystal Structure of Escherichia coli CusC, the Outer Membrane Component of a Heavy Metal Efflux Pump

ABSTRACT VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.

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CusBA Heavy-Metal Efflux Complex of Escherichia Coli

Encyclopedia of Inorganic and Bioinorganic Chemistry ◽

10.1002/9781119951438.eibc2054 ◽

2012 ◽

Author(s):

Chih-Chia Su ◽

Feng Long ◽

Edward W Yu

Keyword(s):

Escherichia Coli ◽

Heavy Metal ◽

Metal Efflux ◽

Heavy Metal Efflux

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Crystallographic Analysis of the CusBA Heavy-Metal Efflux Complex of Escherichia coli

Methods in Molecular Biology - Bacterial Multidrug Exporters ◽

10.1007/978-1-4939-7454-2_4 ◽

2017 ◽

pp. 59-70

Author(s):

Jared A. Delmar ◽

Edward W. Yu

Keyword(s):

Escherichia Coli ◽

Heavy Metal ◽

Crystallographic Analysis ◽

Metal Efflux ◽

Heavy Metal Efflux

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Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export

mBio ◽

10.1128/mbio.00452-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Mitchell A. Moseng ◽

Meinan Lyu ◽

Tanadet Pipatpolkai ◽

Przemyslaw Glaza ◽

Corey C. Emerson ◽

...

Keyword(s):

Heavy Metal ◽

Metal Ion ◽

Efflux Pump ◽

Structural Data ◽

Md Simulations ◽

Efflux Pumps ◽

Content Type ◽

Rnd Efflux Pumps ◽

Metal Efflux ◽

Heavy Metal Efflux

ABSTRACT Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The Escherichia coli CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer. IMPORTANCE The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

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.

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Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei

10.1101/2020.09.18.303271 ◽

2020 ◽

Author(s):

Megan E Grund ◽

Soo J Choi ◽

Dudley H McNitt ◽

Mariette Barbier ◽

Gangqing Hu ◽

...

Keyword(s):

Outer Membrane ◽

Burkholderia Pseudomallei ◽

Triple Helix ◽

Efflux Pump ◽

Fusaric Acid ◽

Efflux Pumps ◽

Resistant Bacteria ◽

Burkholderia Mallei ◽

Membrane Component ◽

Collagen Triple Helix

AbstractBacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the Tier one select agents Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using RT-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the MIC of FA 4-fold in its isogenic mutant. We furthermore showed that the Bucl8 pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8 pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.Author SummaryBurkholderia pseudomallei and Burkholderia mallei are highly infectious and multidrug resistant bacteria that are classified by the National Institute of Allergy and Infectious Diseases as Tier one select agents partly due to the intrinsic multidrug resistance associated with expression of the efflux pumps. To date, only few efflux pumps predicted in Burkholderia spp. have been studied in detail. In the current study we introduce Bucl8, an outer membrane component of an unreported putative efflux pump with a unique extended extracellular portion that forms a collagen triple helix and binds fibrinogen. We demonstrate Bucl8’s role in fusaric acid resistance by defining its operon via bioinformatic and transcriptional analyses, as well as by employing loss-of-function and gain-of-function genetic approaches. Our studies also implicate the Bucl8-associated pump in metabolic and physiologic homeostasis. Understanding how Bucl8 efflux pump contributes to Burkholderia pathology will foster development of pump inhibitors targeting transport mechanism or identifying potential surface-exposed vaccine targets.

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BaeR participates in cephalosporins susceptibility by regulating the expression level of outer membrane proteins in Escherichia coli

The Journal of Biochemistry ◽

10.1093/jb/mvaa100 ◽

2020 ◽

Author(s):

Shuaiyang Wang ◽

Chunbo You ◽

Fareed Qumar Memon ◽

Geyin Zhang ◽

Yawei Sun ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Expression Level ◽

Antibiotics Resistance ◽

Dilution Method ◽

Expression Levels ◽

E Coli

Abstract The two-component system BaeSR participates in antibiotics resistance of Escherichia coli. To know whether the outer membrane proteins involve in the antibiotics resistance mediated by BaeSR, deletion of acrB was constructed and the recombined plasmid p-baeR was introduced into E. coli K12 and K12△acrB. Minimum inhibitory concentrations (MICs) of antibacterial agents were determined by 2-fold broth micro-dilution method. Gene expressions related with major outer membrane proteins and multidrug efflux pump-related genes were determined by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the MICs of K12ΔacrB to the tested drugs except for gentamycin and amikacin decreased 2- to 16.75-folds compared with those of K12. When BaeR was overexpressed, the MICs of K12ΔacrB/p-baeR to ceftiofur and cefotaxime increased 2.5- and 2-fold, respectively, compared with their corresponding that of K12△acrB. At the same time, the expression levels of ompC, ompF, ompW, ompA and ompX showed significant reduction in K12ΔacrB/p-baeR as compared with K12△acrB. Moreover, the expression levels of ompR, marA, rob and tolC also significantly ‘decreased’ in K12ΔacrB/p-baeR. These findings indicated that BaeR overproduction can decrease cephalosporins susceptibility in acrB-free E. coli by decreasing the expression level of outer membrane proteins.

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Yersinia pestis Is Viable with Endotoxin Composed of Only Lipid A

Journal of Bacteriology ◽

10.1128/jb.187.18.6599-6600.2005 ◽

2005 ◽

Vol 187 (18) ◽

pp. 6599-6600 ◽

Cited By ~ 22

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.

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