Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct 9;109(41):16696-701.
doi: 10.1073/pnas.1210093109. Epub 2012 Sep 24.

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

Errett C Hobbs  1 , Xuefeng Yin, Brian J Paul, Jillian L Astarita, Gisela Storz
Affiliations

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

Errett C Hobbs et al. Proc Natl Acad Sci U S A. .

Abstract

The AcrAB-TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli. Here we show that AcrZ (formerly named YbhT), a 49-amino-acid inner membrane protein, associates with the AcrAB-TolC complex. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. The highly conserved acrZ gene is coregulated with acrAB through induction by the MarA, Rob, and SoxS transcription regulators. In addition, mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ may enhance the ability of the AcrAB-TolC pump to export certain classes of substrates.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AcrZ is a highly conserved transmembrane sprotein. (A) acrZ is located between two operons involved in molybdenum metabolism. (B) acrZ is present in virtually all enteric bacteria. The AcrZ protein contains one predicted TM domain at the N-terminal region (13). The sequences shown were aligned using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Arrows indicate positions of two dominant-negative mutations. Based on an alignment with AcrB TM2 (Fig. S3A), the actual TM domain of AcrZ may correspond to amino acids 2–21 instead of 9–29 as predicted.
Fig. 2.
Fig. 2.
AcrZ–SPA interacts with members of the AcrAB–TolC efflux pump. The interacting partners of a functional AcrZ-epitope fusion protein (AcrZ–SPA) were copurified by passing cell lysates over α-FLAG beads and calmodulin beads in a two-step process. Eluates from each column were subjected to SDS/PAGE. Bands enriched in lysates from cells containing AcrZ–SPA were excised from the gel and identified by mass spectrometry.
Fig. 3.
Fig. 3.
AcrZ–SPA associates with AcrAB–TolC through an interaction with AcrB. (A) AcrZ–SPA only bound the Ni2+-NTA resin when hexahistidine-tagged AcrB (AcrB–His6) was present. (B) One dominant-negative form of AcrZ-SPA (G46Stop) associates with AcrB while the other dominant-negative form (G30R) does not. For A and B, cell lysates (L) were passed over Ni2+-NTA resin, and the flow-through (FT) fractions were collected. Bound proteins were washed (W) with a 20-mM imidazole buffer and subsequently eluted (E) from the resin by using a 250-mM imidazole solution. All four fractions were subjected to SDS/PAGE and immunoblot analysis to detect SPA-tagged proteins (α-FLAG) or AcrB (α-AcrB). (C) AcrB–His6 shows a differential trypsin digestion pattern in the presence of AcrZ. AcrB–His6 was purified from wild-type acrZ or ΔacrZ cells and mixed with trypsin (500 mg/mL:1 mg/mL ratio) and incubated at 37 °C for 1, 5, 10, 30, or 60 min. Digestion products were separated on SDS/PAGE and subjected to immunoblot analysis with α-AcrB antibody.
Fig. 4.
Fig. 4.
Suppressor mutations in AcrB restore its interaction with AcrZG30R and resistance to chloramphenicol. (A) AcrBH526Y and AcrBL984P but not wild-type AcrB interact with AcrZG30R in a bacterial two-hybrid assay. β-Galactosidase activity was determined for cells expressing the AcrZ and AcrB proteins fused to the T18 and T25 fragments of B. pertussis adenylyl cyclase, respectively. High β-galactosidase activity indicates efficient reconstitution of enzyme function and affinity between the fused pair of proteins. For each strain, the enzyme activity reported is the average of three independent trials, and the error bars represent 1 SD. The levels of the wild-type and mutant derivatives of the T18 and T25 fusion proteins are comparable (Fig. S2). (B) Chloramphenicol resistance is restored in AcrBH526Y strains expressing AcrZG30R. Overnight cultures mixed as follows were diluted 1:2,000 into liquid LB medium: ΔlacZ/pBAD24 with lacZ+/pBAD24-acrZ, ΔlacZ/pBAD24 with lacZ+/pBAD24-acrZG30R, and acrBH526Y ΔlacZ/pBAD24 with acrBH526Y lacZ+/pBAD24-acrZG30R. The mixed cultures were split, and one-half was treated with chloramphenicol (2 μg/mL). The reported competitive index, which represents the ratio of the tested strain to the reference strain (as measured by colony-forming units) in treated cocultures, normalized to the same ratio in mock treated cocultures (15), is the average of three independent trials, and the error bars represent 1 SD.
Fig. 5.
Fig. 5.
acrZ is regulated by MarA, Rob, and SoxS. (A) A class II mar/rob/sox box is present upstream of acrZ. The invariant adenine at position 1 is highly conserved as is the GCAC “core” of the mar box (Y = C or T, R = G or A, W = A or T, and n = any residue; residues that match the consensus and are conserved in all sequences listed are indicated by capital letters). The 19-bp spacing of the mar/rob/sox box relative to the “−10” region recognized by σ70 is also indicative of a class II promoter structure. (B) Primer extension analysis of total RNA isolated from wild-type and ∆acrZ mutant cells treated with 250 μM paraquat for 10 min shows acrZ induction by paraquat. The sequencing ladder generated with the same labeled oligonucleotide used in the primer extension reactions corresponds to the strand complementary to the ORF. (C) Plasmids (pRGM9817) (20) constitutively expressing marA (pRGM-marA) (20), rob (pRGM-rob) (21), or soxS (pRGM-soxS) (20) were transformed into a strain containing acrZ-SPA. Samples were collected from each strain during the exponential and stationary phases of growth. Lysates were subjected to SDS/PAGE and immunoblot analysis to measure levels of AcrZ–SPA (α-FLAG) and AcrB (α-AcrB).
Fig. 6.
Fig. 6.
ΔacrZ cells are hypersensitive to a subset of antibiotics to which ∆acrB mutants show sensitivity. Overnight cultures of otherwise wild-type ΔlacZ cells were mixed with either wild-type MG1655 cells or ∆acrB or ΔacrZ cells and diluted 1:2,000 in liquid LB medium. These mixed cultures were split, and one-half was treated with tetracycline (0.5 μg/mL), puromycin (45 μg/mL), chloramphenicol (2 μg/mL), erythromycin (60 μg/mL), rifampicin (5 μg/mL), or fusidic acid (60 μg/mL). The reported competitive index is as in Fig. 4. The predicted partition coefficients XlogP3 (PubChem) for the antibiotics are −2, 0, 1.1, 2.7, 4.0, and 5.5, respectively.
See this image and copyright information in PMC

References

    1. Otto M. Looking toward basic science for potential drug discovery targets against community-associated MRSA. Med Res Rev. 2010;30:1–22. - PMC - PubMed
    1. Nikaido H, Pagès JM. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev. 2012;36:340–363. - PMC - PubMed
    1. Grkovic S, Brown MH, Skurray RA. Regulation of bacterial drug export systems. Microbiol Mol Biol Rev. 2002;66:671–701. - PMC - PubMed
    1. Randall LP, Woodward MJ. The multiple antibiotic resistance (mar) locus and its significance. Res Vet Sci. 2002;72:87–93. - PubMed
    1. Blair JM, Piddock LJ. Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: An update. Curr Opin Microbiol. 2009;12:512–519. - PubMed

Publication types

MeSH terms