Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

Abstract

Background: The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production.

Results: In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively).

Conclusions: The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

Figure 1

Alignment of the amino acid sequence of the OtrC-ORF1 region with DrrA and Msr-ORF1. The dark gray regions show residues that are highly conserved, whereas the residues in light gray are less well conserved.

Figure 2

Alignment of the amino acid sequence of predicted conserved motifs of the transmembrane subunit, OtrC-ORF2, MsbA, DrrB, BtuC and MalG. The dark gray regions show residues that are highly conserved, whereas the residues in light gray are less well conserved. The first amino acid in the sequence of each protein is shown.

Figure 3

OtrC heterologous expression in E. coli. Cultures of cells were grown and induced by 1 mM IPTG as described in the Materials and Methods. Cells were resuspended in 500 mM Tris–HCl (pH 7.0) buffer, and cell disruption was performed by ultrasonic waves; the total membrane fractions were harvested by centrifugation at 125,000 rpm at 4°C for 1 h. 12% SDS-PAGE analysis was performed with a high-molecular-weight protein marker (M), 80 μg total membrane fractions of E. coli/pET28a (lane 1), 40 μg, 60 μg and 80 μg total membrane fractions of E. coli/pETC02 (lane 2,3 and 4), 120 μg total cell protein of E. coli/pET28a (lane 5), 60 μg,80 μg and 120 μg total cell protein of E. coli/pETC02 (lanes 6,7 and 8). Western blot analysis was performed with equal volumes of total cell protein of E.coli/pET28a (lane 9) and E. coli/pETC02 (lanes 10, 11 and 12), His-tag mouse and peroxidase-conjugated goat anti-mouse antibodies were used for specific band detection.

Figure 4

ATPase assay of OtrC-overexpressing and OtrC-nonexpressing cells. OtrC from S. rimosus was introduced into the E.coli BL21(DE3) strain using a pETC02 plasmid, and the E.coli BL21(DE3) carrying the empty pET28a was used as the control. The cells were induced by 1 mM IPTG at 30°C for 10 h, and then collected by centrifugation. The cell wall was digested by lysozymes and the membrane vesicles were harvested by centrifugation. The ATPase activity of E.coli/pETC02 (dark gray) was determined compared with to the E. coli/pET28a control (light gray). Vertical error bars corresponded to the standard error of the mean of three replicated samples.

Figure 5

Ethidium bromide (EB) transport in OtrC-overexpressing cells and nonexpressing cells. A: The OtrC-expressing cells (black squares) and the control cells (gray triangles) were cultured and induced with 1 mM IPTG, and then resuspended in KPi buffer (pH 7.0) containing 5 mM MgSO₄. The de-energized cells (OD₆₀₀ = 0.5) were pre-equilibrated with 20 μM EB at 30°C, and 25 mM glucose was added (at the arrow) to initiate EB efflux. B: The effect of orthovanadate on the accumulation of EB in OtrC-expressing and nonexpressing cells. E.coli/pETC02 cells were cultured, induced and collected; the washed cells (OD₆₀₀ = 0.5) were incubated for 10 min at 30°C in the presence (dark gray stars) or absence (black squares) of 0.5 mM orthovanadate, followed by the addition of 20 μM ethidium bromide (EB) along with 2 mM Mg-ATP (arrow). The E. coli/pET28a cells were used as the control, treated with (grey triangles) or without (light gray circles)orthovanadate as described above. The development of fluorescence of the DNA-ethidium complex in the cell suspension was monitored at 30°C every 20 s. The fluorescence intensity before the addition of glucose or ATP was normalized to 100%.

Figure 6

Specific OTC production of S. rimosus strains. A: An extra copy of otrC was introduced into the S. rimosus M4018 chromosome using the recombinant integrative vector pSEC (white circles). The otrC disruption mutant of M4018 was constructed using the temperature-sensitive recombinant plasmid pKCΔotrC (white stars). M4018/pSET152 (black squares) served as the control. B: An extra copy of otrC was introduced into the chromosome of S. rimosus SR16 using the integrative vector pSEC (white diamonds). The otrC disruption mutant of SR16 was constructed using the temperature-sensitive plasmid pKCΔotrC (white hexagons). SR16 that carried the empty pSET152 plasmid was used as the control (black triangles). S. rimosus strains were grown in MS plates, spores were inoculated into GYCS medium to the final concentration of 1 × 10⁶ spores per ml, after which they were cultured at 28°C on a rotary shaker (260 rpm) for 72 h; 1% seed culture was transferred into SC medium cultured at 30°C on a rotary shaker (260 rpm) for 7 d. OTC was measured by HPLC. Vertical error bars correspond to the standard error of the mean of three replicated cultures.

Figure 7

qRT-PCR analysis of the transcriptional level of otrC in S. rimosus mutants. A: The transcriptional level of otrC in M4018/pSEC at 24 h (light gray) and 96 h (dark gray) during OTC fermentation, where M4018/pSET152 was used as the control. B: The transcriptional level of otrC in SR16/pSEC at 24 h (light gray) and 72 h (dark gray) during OTC fermentation, SR16/pSET152 was used as the control. S. rimosus cells were collected at different developmental stages during culture in fermentation medium. RNA extraction and quality control is described in the Materials and Methods. Three housekeeping genes, 16S rRNA, hrdB and zwf1, were used as the reference genes for qRT-PCR analysis.

Figure 8

The relative fold change of MICs of OtrC-expressing cells with different drugs compared to OtrC-nonexpressing cells. A: The relative change in the MIC of OtrC-expressing E. coli/pETC02 cells (gray) to ampicillin (A), rifampicin (R), oxytetracycline (O), doxorubicin (D), ethidium bromide (E), vancomycin (V), ofloxacin (Of), erythromycin (Er) and streptomycin (S), using the OtrC-nonexpressing E.coli/pET28a cells (light gray) as the controls. The relative change in MIC to the drug that could completely inhibit the cell growth of E.coli/pET28a was set as “0”, while the MIC of drug which could not inhibit the cell growth was set as “1”. Cells were grown in LB liquid medium supplemented with kanamycin, IPTG and different concentrations of test drugs at 37°C, and spun at 170 rpm for 12 h. Cells were then resuspended in fresh LB and spread immediately on the LB plates with kanamycin, IPTG and different concentration of the test drugs. The MICs to drugs were determined after the plates were cultured at 37°C for 12 h. B: The relative fold of MICs of OtrC-expressing S. rimosus mutants, M4018/pSET152 (light gray) and M4018/pSEC (gray), to the above-mentioned drugs, using the OtrC-nonexpressing mutant M4018/pKCΔotrC (dark gray) as the control. The relative change in the MIC to drugs that could completely inhibit the cell growth of M4018/pKCΔotrC was set as “0”, while the MIC of drug which could not inhibit the cell growth was set as “1”. Cells were grown in TSB liquid medium supplemented with different concentrations of drugs at 28°C, and the centrifuged at 220 rpm for 30 h, resuspended in fresh TSB and spread immediately on TSB plates with different concentrations of test drugs. The MICs of drugs were determined after the plates were cultured at 28°C for 3 d. ** indicate significantly higher MIC values than the control cells; * indicate higher MIC values than the control cells.