An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump

Abdelmotaal, Heba; Meng, Lin; Zhang, Zhenglai; Abdelazez, Amro; Shao, Li; Xu, Tong; Meng, Fankui; Abozaed, Shaima; Zhang, Rui; Jiang, Juquan

doi:10.3389/fmicb.2018.01601

Cited by 21 publications

(34 citation statements)

References 68 publications

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“…Accumulation of norfloxacin in E. coli CM2/pET-mdrP was found to be significantly enhanced by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton gradient uncoupler. However, accumulation of norfloxacin in E. coli CM2/pET-mdrP was unable to reach the same maximum level as that of CM2/pET19 after the addition of CCCP (Abdel-Motaal et al, 2018). Considering MdrP being a Na + /H + antiporter, we hypothesize that MdrP may bind with Na + and therefore employ it as a coupling ion to drive norfloxacin efflux in a mode of drug/Na + antiport, similarly to those of Na + -coupled MATE transporters, N. gonorrhoeae NorM (Lu et al, 2013b) and V. cholerae NorM (Jin et al, 2014).…”

Section: Introductionmentioning

confidence: 90%

“…In our recent study (Abdel-Motaal et al, 2018), an uncharacterized MFS transporter from Planococcus maritimus has been identified to function as a Na + (Li + , K + )/H + antiporter and also as a multidrug efflux transporter with the resistance against multiple antimicrobial drugs, such as norfloxacin (Abdel-Motaal et al, 2018). In addition, this transporter was suggested to maybe belong to DHA1 family within MFS (Abdel-Motaal et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux

et al. 2020

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Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na +coupled MFS-MDR transporter, MdrP, which also can achieve H +-coupled drug efflux independently of Na +. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na + antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na + and H + coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na + translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na + and H + coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.

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Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux

et al. 2020

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“…Purchased from Takara and then their growth was determined. The growth assay was conducted according to the protocol described by previous reports (Meng et al, 2017;Wang et al, 2017;Abdel-Motaal et al, 2018).…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

A Putative Efflux Transporter of the ABC Family, YbhFSR, in Escherichia coli Functions in Tetracycline Efflux and Na+(Li+)/H+ Transport

et al. 2020

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ATP-binding cassette transporters are ubiquitous in almost all organisms. The Escherichia coli genome is predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters, of which seven are possible drug export transporters. There has been minimal research on the function of YbhFSR, which is one of the putative drug resistance exporters. In this study, the ybhF gene of this transporter was characterized. Overexpression and knockout strains of ybhF were constructed. The ATPase activity of YbhF was studied using the malachite green assay, and the efflux abilities of knockout strains were demonstrated by using ethidium bromide (EB) as a substrate. The substrates of YbhFSR efflux, examined with the minimum inhibitory concentration (MIC), were determined to be tetracycline, oxytetracycline, chlortetracycline, doxycycline, EB, and Hoechst33342. Furthermore, tetracycline and EB efflux and accumulation experiments confirmed that the substrates of YbhFSR were tetracyclines and EB. The MIC assay and the fluorescence test results showed that tetracyclines are likely to be the major antibiotic substrate of YbhFSR. The existence of the signature NatA motif suggested that YbhFSR may also function as a Na + /H + transporter. Overexpression of YbhF in E. coli KNabc lacking crucial Na + /H + transporters conferred tolerance to NaCl, LiCl, and an alkaline pH. Together, the results showed that YbhFSR exhibited dual functions as a drug efflux pump and a Na + (Li +)/H + antiporter.

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“…Excitation wavelength was 500 nm (9-nm slit), and emission wavelength was 580 nm (20-nm slit). The EB efflux tests were performed according to the protocol as described by Abdel-Motaal et al [29], Reuter et al [8]and Balakrishnan et al [30].…”

Section: Fluorescence Testsmentioning

confidence: 99%

“…The excitation and emission wavelengths were 277 and 448 nm for norfloxacin. The norfloxacin accumulation test was performed as described by Abdel-Motaal et al [29].…”

Section: Fluorescence Testsmentioning

confidence: 99%

Cloning and Functional Characterization of Putative Escherichia coli ABC Multidrug Efflux Transporter YddA

Feng¹,

Liu²,

Liu³

et al. 2020

J. Microbiol. Biotechnol.

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/nodulation/cell division (RND), the proteobacterial antimicrobial compound efflux (PACE) family and the p-aminobenzoyl-glutamate transporter (AbgT) family [1]. Of these, SMR, MFS, MATE, RND, PACE and AbgT use proton kinetic potential as energy and ABC transporters use ATP as energy. All of them can export drugs and other substrates out of cells [2]. Some proteins, although they confer low levels of resistance, are often the first step in resistance, eventually leading to higher resistance by acquiring chromosomal mutations that target antibiotics. The ABC transporter is one of these proteins [3]. ABC transporters exist in almost all organisms from microorganisms to humans [4, 5]. ABC transporters have attracted extensive attention because of their involvement in important physiological processes such as bacterial resistance, human cystic fibrosis, and tumor cell resistance to chemotherapy drugs [6]. ABC transporters contain similar topologies, with two transmembrane domains (TMD) and two nucleotide-binding domains (NBD). These four domains can fuse to form a full transporter such as the multidrug efflux pump P-glycoprotein (P-gp, ABCB1 or MDR1) in humans. In bacteria, multidrug efflux pumps usually fuse to form a half-transporter with a TMD and an NBD domain [7]. The half-transporter forms a homodimer such as MsbA [7] or LmrA [8]. It can also form heterodimers such as EfrCD [9], YheI/YheH [10] and PatA/PatB [11]. The E. coli K-12MG1655 strain genome was predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters [5, 12]. Efflux systems of the ABC family in E. coli were reported to mainly include capsular polysaccharide transport pump KpsT [13], hemolysin transport pump HlyA [14, 15], macrocyclic lipid drug efflux pump MacAB [16], outer membrane lipoprotein transport pump LolACDE [17-19], multidrug resistance and phospholipid A transporter MsbA [20], micromycin J25 efflux pump YojI [21], and heme efflux pump CcmABC [22]. Drug efflux systems in E. coli that have been reported included MsbA and MacA. MsbA is a multidrug efflux pump and MacAB is a single-drug efflux pump of macrolides[16]. Studies reported on the A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drugsensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norf...

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An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump

Cited by 21 publications

References 68 publications

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux

A Putative Efflux Transporter of the ABC Family, YbhFSR, in Escherichia coli Functions in Tetracycline Efflux and Na+(Li+)/H+ Transport

Cloning and Functional Characterization of Putative Escherichia coli ABC Multidrug Efflux Transporter YddA

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