Bacterial strains, plasmids, antibiotics and antibodies
The bacterial strains used in this study were E. coli DH5α, ET12567 (pUZ8002)
, BL21 (DE3), S. rimosus M4018 (a typical strain, gift from Prof. Iain S Hunter, University of Strathclyde, UK), and SR16 (an industrial overproducer obtained by traditional strain improvement, Shanxi Tongxing Antibiotic Company, China).
The plasmids used in this study included pMD19-T, pSET152, pKC1139, pET28a and pSEC (otrC in pSET152), pKCΔotrC (for otrC disruption, reconstructed by pKC1139), pETC02 (otrC in pET28a). E. coli strains were screened using a LB plate with 100 μg·ml-1 ampicillin, 50 μg·ml-1 apramycin, 25 μg·ml-1 kanamycin or 34 μg·ml-1 chloramphenicol when appropriate. S. rimosus mutants were screened by TSB plates with 500 μg·ml-1apramycin or 500 μg·ml-1 kanamycin.
Different concentration of ampicillin, oxytetracycline, doxorubicin, ethidium bromide, vancomycin, ofloxacin, rifampicin, erythromycin and streptomycin were used for the MDR assay with OtrC. His-tag mouse and peroxidase-conjugated goat anti-mouse IgG (SAB, USA) antibodies were employed for Western blotting analyses.
Media and growth conditions
Escherichia coli strains were cultured in Luria-Bertani (LB) medium (1.0% tryptone, 1.0% NaCl, 0.5% yeast extract) with appropriate antibiotics when necessary. S. rimosus strains were grown in tryptone soya broth (TSB) medium (3% tryptone soya broth, Oxoid) for genomic DNA extraction and mycelium growth, adding appropriate antibiotics, where needed. The mannitol soya flour (MS) medium (2% mannitol, 2% soya flour and 2% agar) was used to grow spores
. The seed cultures were obtained by inoculating spores into glucose yeast casein hydrolysate and sucrose (GYCS) medium (1% glucose, 0.05% yeast extract, 1.5% casein hydrolysate, 0.28% sucrose, 0.01% CaCO3). Soluble starch and corn steep liquor (SC) medium
 (2% soluble starch, 1% corn steep liquor, 0.6 (NH4)2SO4, 0.8% CaCO3, 0.5% NaCl, 0.2% soy bean oil, pH 6.8 to 7.2).
OtrC expression in E. coli
For OtrC heterologous expression in E. coli, the genomic DNA of M4018 was used as the template. The following primers, orf1F (5’CGCCATATGATGAC GCGAAAGACGATATCCA3’) and orf1R (5’CGCGGATCCTCATGCCGGAACCTCCTCG3’), were used to amplify the otrC open read frame 1 (orf1), which was introduced into the NdeI and BamHI sites (underlined); primers orf2F (5’CGCGGATCC
GAAGGAGATATACCATGAGTGCCGCGACGGT3’) and orf2R (5’ATTTGCGGCCGCGGTCTTCTTGCGGAACTTGGC 3’) were used to amplify otrCorf2 which was introduced into the BamHI and NotI sites (underlined), and the optimized RBS sites of the T7 promoter (bold) was introduced towards the 5’ end. The amplified fragments (orf1, 1,074 bp and orf2, 870 bp) were subcloned into T-vector for sequencing, and then digested by restriction enzymes and cloned into the pET28a vector under the T7 promoter to construct the recombinant plasmid, pET28a-otrCorf1-otrCorf2 (pETC02).
After pETC02 was introduced into the E. coli BL21 (DE3) strain, for OtrC expression, the E. coli transformant (E.coli/pETC02) was cultured in LB medium with 50 μg/ml kanamycin. The growth of the cultures was monitored by recording the optical density at 600 nm (OD600); when it reached at 0.6, cells were induced by isopropyl beta-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 30°C, at 170 rpm for 10 h. The E. coli/pET28 transformant was cultured and induced under the same conditions and used as a negative control.
One-milliliter cell suspensions were centrifuged at 12,000 rpm for 10 min at 4°C, and washed with deionized water, then resuspended in 100 μl Tris–HCl (500 mM pH 7.0) buffer, and the cell disruption was performed by ultrasonic waves. The cell disruption suspension was centrifuged at 12,000 rpm for 15 min at 4°C to remove the cell debris, and the total membrane fractions were then harvested by centrifugation at 125,000 rpm, at 4°C for 1 h. The total protein sample and the membrane fractions were then analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting following standard procedures
ATPase assay of OtrC
For the ATPase assay, E. coli transformants were cultured in LB with 50 μg/ml kanamycin and induced with IPTG, as described above. A cell suspension sample of 1.5 ml with OD600 = 0.6 was obtained, and cells were collected by centrifugation at 10,000 rpm for 1 min. Cells were then washed with deionized water twice. Cell walls were digested by incubation for 1 h at 30°C with 10 mg/ml lysozyme (BBI, UK), after which DNase (100 μg/ml, TaKaRa, Japan) and 10 mM MgSO4 were then added. After a 10-min incubation, the suspension was centrifuged at 16,000 rpm for 15 min at 4°C to remove the cellular debris, and the membrane vesicles were then harvested by centrifugation at 125,000 rpm at 4°C for 1 h, and resuspended in 1.5 ml buffer A (50 mM Hepes pH 8.0, 250 mM NaCl, 10% glycerol [v/v]), and used as the protein sample in immediate assays for ATPase activity
The malachite green assay was used to determine the specific ATPase activity of OtrC by measuring the release of inorganic phosphate. Briefly, 2 mM ATP was added into a 200 μl protein sample, 25-μl aliquots were transferred into 175 μl of 10 mM sulfuric acid to stop the reaction for 20 min. Subsequently, 50 μl of fresh malachite green/molybdate solution was added, and the absorbance at 650 nm was measured after incubation for 10–15 min
The malachite green/molybdate solution was freshly prepared and it contained malachite green solutions 1 (0.122% [w/v] malachite green, 20% [v/v] sulfuric acid), solutions 2 (7.5% [w/v] ammonium molybdate) and solutions 3 (11% [v/v] Tween 20) at a ratio of 50:12.5:1
Efflux assay of OtrC
The E. coli/pETC02 was cultured in LB with 50 μg/ml kanamycin and induced with IPTG. Subsequently, the cells were collected by centrifugation at 10,000 rpm at 4°C for 10 min, before being washed with 1 volume 50 mM KPi (pH 7.0) containing 5 mM MgSO4. The washed cell suspensions (OD600 = 0.5) were incubated for 10 min at 30°C in the presence of 20 μM ethidium bromide (EB). The EB efflux was initiated by the addition of 25 mM glucose, and the cell suspension was used to monitor the fluorescence of DNA-EB complex by a fluorimeter (Cary Eclipse, Australia) at 30°C using excitation and emission wavelengths of 500 and 580 nm, respectively, and slit widths of 5 and 10 nm, respectively
To study the effect of ABC inhibitor Ortho-vanadate on the accumulation of EB in OtrC expressing and non-expressing E. coli cells
, cells were cultured, induced, collected and washed as described above. Washed cells (OD600 = 0.5) were incubated for 10 min at 30°C in the presence or absence of 0.5 mM orthovanadate, followed by the addition of 20 μM ethidium bromide (EB) along with 2 mM Mg-ATP, and the fluorescence of EB was measured at 30°C as described above.
OtrC knock-in and disruption in S. rimosus
The ermE*p promoter region was amplified by using E*pf (5’CCGGAATTGTACCAGCCCGACCCGAG3’) and E*pr (5’ CGCGGATCCGTGGAGTGGTTCTGTATCCTACCAA 3’) as the primers to amplify a 306 bp fragment encompassing the EcoRI and BamHI cloning sites. The entire otrC gene (1,895 bp) encompassing the BamHI and XbaI sites were amplified using genomic DNA of M4018 as templates and otrCf (5’ CGCGGATCC
CCTCTTACGAGNAAGTCATGAAGTTCCGCCGAATGNA 3’,) and otrCr (5’ TGCTCTAGATCAGGTCTTCTTGCGG AACTT3’) primers, where the natural RBS sequence of otrC was introduced into primer otrCf (bold). The fragments were cloned into a pMD19-T vector for sequencing, and the fragments were digested by the restriction enzymes after identification and inserted into a pSET152 vector to obtain the recombinant plasmid, pSET152-ermE*p-otrC (pSEC). This was identified by enzyme digesting and sequencing. Plasmid pSEC was introduced into E. coli ET12567 (pUZ8002) for demethylation and then introduced into the chromosomes of M4018 and SR16 by electroporation following standard procedures to generate the M4018/pSEC and SR16/pSEC mutant strains. The empty plasmid, pSET152, was introduced into M4018 and SR16 to construct M4018/pSET152 and SR16/pSET152, respectively, which were used as controls.
For the disruption of otrC, the CZ1 and CZ2 fragments were amplified by PCR using two sets of primer pairs: CZ1f (5’CCGGAATTCTGCCTGCCCGCCGTC3’) and CZ1r (5’ CCGGATATCTCCTCGTGGTCGGCGGT 3’), CZ2f (5’ CGCGGATCCCGGCGTGGTCAACGTC 3’) and CZ2r (5’TGCTCTAGACCCTGTCCGTTCATCNNN 3’), respectively. The kanamycin resistance cassette (Kanr) was amplified by PCR using the primer pairs kanf (5’ CCGGATATCTACAAGGGGTGTTATGAGCC 3’) and kanr (5’ CGCGGATCCTTAGAAAAACTCATCGAGCAT 3’). The final PCR products (CZ
: 993 bp, CZ
: 992 bp, Kanr: 832 bp) were cloned into the pMD19-T vector, and used for sequencing to confirm the correct amplification. Fragments CZ1 and CZ2 were used as the left bridge and the right bridge, respectively. Three resulting DNA fragments were digested by the restriction enzymes and inserted into the EcoRV and XbaI sites of the pKC1139 vector to generate pKC1139-CZ1-Kanr-CZ2 (pKCΔotrC). The recombinant plasmid was demethylated and introduced into either M4018 or SR16. The screening of the otrC disruption mutants M4018/pKCΔotrC and SR16/pKCΔotrC followed standard procedures
. Confirmation of the otrC disruption was performed by the PCR amplification of kanr using genomic DNA of the mutants as a template.
Effect of OtrC expression on drug susceptibility
E.coli/pETC02 was grown in LB liquid medium with 50 μg/ml kanamycin, IPTG (1 mM) and different concentrations of drugs at 37°C, 170 rpm for 12 h, and then resuspended in 1 volume of fresh LB. Then, 50 μl cell suspension was spread immediately on the LB plates with 50 μg/ml kanamycin and 1 mM IPTG and different concentration of drugs, the plates were cultured at 37°C for 12 h to determine the minimal inhibitory concentrations (MICs). Experiments were performed in triplicate using E. coli/pET28a as the negative control.
S. rimosus mutants M4018/pSEC and M4018/pKCΔotrC were grown in TSB adding different concentrations of tested drugs with M4018/pSET152 at 28°C and 220 rpm; after 30 h growth, cells were collected by centrifugation and resuspended in 1 volume of TSB. Then, 50 μl of fresh mycelium suspension was spread immediately on the TSB plates with different concentration of tested drugs. The MICs were measured after incubation for 3 d at 28°C, and experiments were performed in triplicate.
Effect of OtrC expression on OTC production in S .rimosus
The mutants of M4018 and SR16 were cultured on the MS plates at 28°C for 3–5 d along with the parental strains; spores were collected and inoculated into GYCS medium to a final concentration of 1 × 106 spores per ml, after they were cultured at 28°C on a rotary shaker (260 rpm) for 72 h as the seed culture. For an assessment of OTC production, 1% seed culture was transferred into SC medium and cultured at 30°C on a rotary shaker (260 rpm) for 7 d. The OTC productivity of S. rimosus strains were measured by high performance liquid chromatography analysis (HPLC). Then, 1 ml culture samples were centrifuged at 12,000 rpm for 10 min after the pH was adjusted to pH 1.5-1.7 using 9 mol/L HCl. The supernatants were filtered through a 0.22-μm filter (Millipore, Bedford, MA) and 10 μl samples were injected for analysis. Agilent 1100 HPLC system equipped with a 5-μm C18 (Kromasil, Sweden) column (4.6 by 200 nm) was employed for the HPLC analysis. The mixture of 60% H2O, 10% methanol, 20% acetonitrile and 10% phosphoric acid (2 mM) was used for the mobile phase and applied with a constant flow rate of 0.8 ml min-1 over 10 min. The OTC detection wavelength was 350 nm
qRT-PCR analysis of transcription levels
The transcription level of the otrC gene was analyzed by qRT-PCR as described previously
. S. rimosus cells were collected at different developmental stages during fermentation. Fresh tissues were used for total RNA extraction immediately using the AxyPrepTM multisource total RNA miniprep kit (Axygen, USA). The genomic DNA was removed by DNase I digestion, and the purity of the RNA samples was measured as the ratio of RNA concentration (ng/μl) to protein concentration (ng/μl) by QubitTM RNA Assay Kits and QubitTM Protein Assay Kits (Invitrogen, CA, USA), using the Qubit® 2.0 fluorometer (Invitrogen), according to the instructions provided by the manufacturer. RNA samples (RNA:protein ratio, 1.8-2.0) were selected and examined by 3% agarose gel electrophoresis to check their integrity prior to qRT-PCR analysis
[32, 33]. First strand cDNA synthesis was performed with a reverse transcription kit (TaKaRa, Japan) according to the manufacturer’s instructions. Standard curve was consisting of a 10-fold serial dilution series of five points which was prepared from cDNA samples (35 ng/μl) and each dilution was tested under a range of temperatures around the calculated Tm of the primers used in the experiments; untranscribed RNA samples were used as negative controls. The Cq values were determined for the optimization of qRT-PCR conditions.
qRT-PCR was assessed using SYBR® GC Premix Ex TaqTM kits (TaKaRa, Japan) and a CFX96TM 168 real-time PCR detection system (Bio-Rad, USA). The total 25 μl reaction volume contained 1 μl DNA, 0.2 μM forward primer, 0.2 μM reverse primer, and 1× SYBR® Premix Ex TaqTM, and the reaction conditions were as follows: 95°C for 30 s, and 40 cycles of 95°C for 5 s, 55-61°C for 30 s and 72°C for 30 s. Analysis of the melting curve was performed over a range of 55°C to 95°C for 5 s at the end of the PCR cycles. Three housekeeping genes, 16S rRNA, hrdB and G6PDH (zwf1) were used as reference genes to normalize the data
[32, 34]. The optimized Tm and cDNA concentration of each gene were used for qRT-PCR analysis. The primers for qRT-PCR were as follows: QotrCf (GTCACACGAGCGCCCTGGT) and QotrCr (CGCCGCCGAAGACGTACAC) were used for otrC transcriptional level analysis; QhrdBf (CTCTGTCATGGCGCTCA) and QhrdBr (ACGTTCTTCCACTGAGTGG) for the reference gene hrdB; Q16Sf (AGACACGGCCCAGACTC) and Q16Sr (CTGCTGAAAGAGGTTTACAAC) for 16S rRNA; Qzwff (ACTGGGCCAGAACGCCCT) and Qzwfr (AGTCCATCGAGACGTCCCGTA) for zwf1.