Trinity revealed: Stoichiometric complex assembly of a bacterial multidrug efflux pump

Pos, Klaas M.

doi:10.1073/pnas.0902837106

Cited by 17 publications

(11 citation statements)

References 21 publications

(24 reference statements)

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“…A minimum of five shoot tips were inoculated for each strain. Data represent the numbers of CFU/cm of stem/shoot tip times 10 7 . Average values and standard deviations are listed in Table S5 in the supplemental material.…”

Section: Figmentioning

confidence: 99%

“…In Gram-negative bacteria, efflux pumps that form a large tripartite complex spanning both the inner and outer membranes play a key role in multidrug resistance (3)(4)(5). These pumps consist of an inner membrane transporter (ABC, MFS, or RND family transporter), a membrane fusion protein, and an outer membrane channel (6,7). In E. amylovora, the RND-type efflux pump AcrAB-TolC has been demonstrated to play an important role in virulence (8,9).…”

mentioning

confidence: 99%

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AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

2014

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bTranscriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/ XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses.

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Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

2014

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“…The question arises whether or not there is a functional meaning to the existence of such a hydrophobic patch on the periplasmic domain of AcrB. Although the current model of the AcrABTolC complex (Symmons et al, 2009) suggests that the DARPin binding site would not be involved in the formation of the functional assembly (Pos, 2009), we suggest that this area is part of the interaction surface of AcrB and AcrA. However, further experimental evidence is needed to clarify this issue.…”

Section: Dominant Epitopementioning

confidence: 91%

Designed ankyrin repeat protein binders for the crystallization of AcrB: Plasticity of the dominant interface

Monroe

Sennhauser

Seeger

et al. 2011

Journal of Structural Biology

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“…RND proteins are known to have the ability to form tripartite efflux complexes that span both the inner and outer membranes of Gram-negative bacteria. These protein complexes consist of the RND protein, a pore forming outer membrane protein (OMP) and a membrane fusion protein (MFP) [Pos, 2009]. The ORF of A1S_3445/6 is preceded by a gene encoding a predicted MFP and a divergently transcribed regulator.…”

Section: Annotation and Isolation Of The A Baumannii Efflux Systemsmentioning

confidence: 99%

Development of a High-Throughput Cloning Strategy for Characterization of Acinetobacter baumannii Drug Transporter Proteins

et al. 2011

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Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.

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Trinity revealed: Stoichiometric complex assembly of a bacterial multidrug efflux pump

Cited by 17 publications

References 21 publications

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

Designed ankyrin repeat protein binders for the crystallization of AcrB: Plasticity of the dominant interface

Development of a High-Throughput Cloning Strategy for Characterization of Acinetobacter baumannii Drug Transporter Proteins

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