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

Abstract

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.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“… 538 This tripartite system can efflux cephalosporins, including the third generation cefoperazone. Recently, Feng et al 62 characterized the YbhFSR system and found it to export tetracycline, oxytetracycline, chlortetracycline, doxycycline, EtBr, as well as the Hoechst 33342 dye. Furthermore, uniquely for the ABC-family, the findings of Feng et al 62 suggested that YbhFSR exhibits a dual function both as a MDR efflux pump and as a Na + (Li + )/H + antiporter, although this requires further investigation, especially in light of the rather high homology of the YbhFSR protomers with MATE transporters such as NorM.…”

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

“…For example, Hoechst was used in studies of drug efflux-based antibiotic resistance in Salmonella enterica serovar Typhimurium 8 , 9 and Acinetobacter baumannii 10 , 11 , and in transport measurements for the ABC multidrug transporters LmrA from Lactococcus. lactis 12 – 14 , Sav1866 from Staphylococcus aureus 15 , MsbA and YbhFSR from E. coli 16 , 17 , and PatAB from Streptococcus pneumoniae 18 . Hoechst transport assays were used in studies of secondary-active multidrug and toxic compound extrusion (MATE) transporters VcmA from Vibrio cholerae 19 and AbeM from A. baumannii 20 , and in studies of a novel natural product inhibitor of the major facilitator superfamily (MFS) multidrug transporter NorA from Staphylococcus aureus 21 .…”

The worm affair: fidelity and environmental adaptation in symbiont species that co‐occur in vestimentiferan tubeworms

“…The presence of an adjacent phage integrase indicates the potential for acquisition of the ATPase and ABC transporter via horizontal gene transfer. In Escherichia coli , Ybh transporters excrete tetracyclines, but also confer tolerance to NaCl, LiCl and an alkaline pH (Feng et al ., 2020), and thus may allow adaptation for specific physiological conditions. Only species B symbionts carried a gene cluster involved in fatty acid metabolism, including genes that encoded (in this order) very long‐chain acyl‐CoA dehydrogenase, 3‐ketoacyl‐CoA thiolase and 3‐hydroxyacyl‐CoA dehydrogenase, as well as FixAB electron transfer flavoprotein, indicating the ability to use alternative carbon sources (Srivastava et al ., 2015; Menendez‐Bravo et al ., 2017).…”