The bacteria and plasmids used in this study are listed in Table 1. E. coli was cultured by incubation at 37 °C. At the same time, P. protegens was cultured at 28 °C in lysogeny broth (LB; solid medium plus 1.5% agar). The antibiotics and concentrations used for P. protegens and E. coli culture were as follows: 40 μg/mL of kanamycin, 50 μg/mL of gentamicin, and 100 μg/mL of ampicillin. The concentration of sucrose used was 10% (w/v) when gene knockout was performed using the suicide plasmid pJQ200SK. Other components were isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM), ortho-nitrophenyl-β-D-galactopyranoside (4 mg/mL) and Taqaq (TaKaRa, Shiga, Japan). DNA ligase, plasmid preparation, restriction endonucleases, RNA reverse transcriptase, DNA gel extraction, and KOD Plus DNA polymerase (TaKaRa) were performed based on manufacturer’s protocol described in the commercial kits (Omega Bio-Tek, Doraville, GA, USA). Primers (synthetic oligonucleotides) were purchased from Anygene Biological Technology Co., Ltd. (Wuhan, China). Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China) provided DNA sequencing services. All molecular biology procedures were used based on standard methods.
The rsmY genes (900-bp and-800-bp, respectively) and the upstream and downstream fragments of the hfq (1000-bp and 900-bp, respectively) were fused by polymerase chain reaction (PCR) and digested with XbaI/HindIII along with the suicide plasmid pJQ200SK prior to their ligation to and construction of the vectors pJQΔhfq and pJQΔrsmY. The knockout vectors pJQΔhfq and pJQΔrsmY were then transferred into P. protegens Pf-5, and mutants were selected on 10% sucrose LB plates. The P. protegens Pf-5 harboring plasmid pJQ200SK was unable to grow on 10% sucrose plates, indicating that the double-recombination of the strains resulted in loss of plasmid pJQ200SK. Polymerase chain reaction (PCR) and sequencing confirmed the knockout of hfq and rsmY genes, and the strains were named PfΔhfq and PfΔrsmY, respectively. The hfq and rsmY double-gene knockout was achieved using the same methods. The knockout vector pJQΔrsmY was transferred into PfΔhfq, followed by selection for the rsmY-knockout mutant, and the double-gene knockout strain was named pfΔhfqΔrsmY. Here, pRK2073 was used as a helper plasmid, which was transferred into P. protegens Pf-5 using tri-parental hybridization.
The recombinant-expression plasmid pBBR-hfq was created to construct an hfq complementation strain following knockout. pBBR-hfq was constructed by ligating the promoter sequence and the 547-bp sequence containing the hfq gene into the shuttle plasmid pBBR1MCS-5 of Pseudomonas–E. coli following BamHI/HindIII digestion. Then pBBR-hfq was transferred into the PfΔhfq strain to generate the complementation strain pfΔhfq/pBBR-hfq. The plasmid pBBRK-hfq was constructed by ligating the 547-bp sequence containing the hfq gene and the promoter sequence into plasmid pBBRKm following BamHI/HindIII digestion. The same methods were then used to construct pBBR-rsmX, pBBR-rsmY, pBBR-rsmZ, pBBRK-rsmX, pBBRK-rsmY, and pBBRK-rsmZ.
The promoter-lacZ reporter gene was constructed for studying the regulation of lipase gene expression by hfq, by fusing the lipA promoter sequence with the lacZ sequence. PCR was used to amplify lacZ from the genomic DNA of E. coli BL21 (DE3), with the ‘lacZ amplicon (bp 22–3110 from the start site of translation) lacking the first seven codons and the sequence of Shine–Dalgarno (SD), whereas the amplicon of wild-type lacZ (bp 18–3110 from the start site of translation) contained the SD sequence. Into the plasmid pBBR1MCS-5, lacZ and ‘lacZ were inserted HindIII and BamHI cleavages, and cloned to generate the transcriptional-fusion plasmid pBBR02 and the translational-fusion plasmid PBBR01, respectively. The lipA gene was amplified using PCR, and following KpnI and HindIII cleavages, the lipA amplicons (bp 613–18 from the start site of translation) were cloned into plasmid pBBR01 and cloned to generate plasmid pBBR03. Similarly, the inserted lipA′ amplicons (bp 613–12 from the start site of translation) in plasmid pBBR02 generated plasmid pBBR04 (Table 1). Into plasmid pJQ200SK, lipA′–‘lacZ and lipA-lacZ were inserted following pBBR03 and pBBR04 cleavages with BamHI and SphI, and cloned to generate plasmids pJQ003 and pJQ004, respectively. The same methods were utilized to construct rsmZ’-‘lacZ, rsmY’-‘lacZ, rsmE’-‘lacZ, rsmA’-‘lacZ, and phrs’-‘lacZ.
P. protegens Pf-5 was cultured until the level of growth attained optical density value of about 5.5 at 600 nm (OD600) and thereafter, RNA was extracted using RNA extraction kit (CWBIO, Beijing, China). Following purification, 2 μg of the RNA was reverse-transcribed using random hexamer primers as described in Revert Aid kit instruction leaflet for first-strand cDNA synthesis (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR machine (ABI 7500; Applied Biosystems, Foster City, CA, USA) was employed for quantitative RT-PCR (qRT-PCR) in 96-well plate with its default program (2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles at 94 °C for 15 s and at 60 °C for 60 s). A total of 20 μL reaction mixture volume was used. The reaction mixture contained 6.4 μL of RNase-free water, 10 ng of final cDNA, 10 pM of each primer, and SYBR Green master mix (10 μL; Roche, Basel, Switzerland). There was a control with an aliquot of RNase-free water of 2.0 μL in each plate. Each plate contained three technical replicates. Prior to qRT-PCR evaluation of the P. protegens Pf-5 genes, PCR-efficiency curves as well as specific verification of the dissociated PCR-amplified candidate reference gene were determined. Using rpoD as an internal reference, differences in mRNA expression were determined.
The 305-bp DNA fragment containing the entire hfq open reading frame sequence (261-bp) was amplified by PCR using P. protegens Pf-5 as a template. After cleavage with the restriction enzymes NdeI/HindIII, the generated fragment was inserted in the expression vector pET28a to produce the hfq-expression vector pET28a-hfq (Table 1). Into E. coli BL21 (DE3) cells, pET28a-hfq was transferred and the host cultured at 37 °C in LB containing 0.5 mM IPTG. Each E. coli BL21 (DE3) was allowed to grow and attain an OD600 of ~0.8. Then it was incubated for 20 h at 16 °C. The cells were thereafter pelleted by centrifugation and re-suspended in nickel A buffer [25 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole] supplemented with 50 μM phenylmethyl sulfonyl, 1 μg/mL aprotinin, and 1 μg/mL leupeptin. After shaking the suspension for 30 min at 4 °C, an ultrasonic cell disruptor was used to lyse the cells. The lysate was allowed to percolate completely into a column of nickel-nitrilotriacetic acid agarose (GE Healthcare, Pittsburgh, PA, USA). The column was washed twice with 5 mL portions of nickel eluting buffer containing 500 mM imidazole, to elute the hfq protein. The purified hfq protein was then stored in a buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 200 mM NaCl and 20 mM Tris–HCl.
Light-shift chemiluminescent RNA EMSA kit (Thermo Fisher Scientific), REMSA was used for REMSA. RNA fragments of rsmA, rsmE, rsmY, rsmZ were synthesized in vitro and labeled with biotin. A 2-μL probe solution containing the respective biotin-labeled RNA fragment was mixed with 3 μL of purified hfq protein (1 mg/mL) in 10 μL of binding buffer [10 mM DTT, 10 mM MgCl2, 200 mM KCl, and 100 mM HEPES, pH 7.3], and placed for 10 min at room temperature to prevent non-specific binding of the protein and probe. In studies on competitive binding, unlabeled probe concentrations were 50-fold, 100-fold, and 150-fold higher than that of the labeled-probe. Binding buffer (1 μL; colorless; 10 × ) was added and mixed immediately. Pre-electrophoresis was conducted for 30 min with 0.5 × TBE (Tris/borate/EDTA) as the electrophoretic solution at 80 V. The electrophoretically-separated protein-RNA conjugates were bound to a positively-charged nylon membrane (Ambion; Thermo Fisher Scientific). The membrane was cross-linked by the free RNAs and transferred hfq-RNA complexes when exposed to UV light at 320 nm. The biotin-labeled nucleic acid bands on the membrane were detected by chemiluminescence (Thermo Fisher Scientific). While on nylon membrane, the transferred biotin-labeled RNAs were visualized by using the activated conjugate of stabilized streptavidin and horseradish peroxidase (HRP). In order to produce light of high sensitivity, the HRP was allowed to act on luminol-based substrate. The luminescent membrane was exposed to X-ray film after remaining in a film cassette for 20–30 seconds.
The strains P. protegens Pf-5 and PfΔhfq were used to determine the stability and steady-state level of rsmY. It was added at an OD600 value of 4.0 to 500 µg/mL of rifampicin (final concentration). Rifampicin was also added to the total RNA isolated from 4 ml aliquot at 0, 10, 20, 30, 40, and 60 min. Aliquots (4-mL portions) were withdrawn. With 2 µg of total RNA, primer extension technique was used to determine rsmY concentrations with AMV reverse transcriptase (Promega, Durham, NC, USA).
Denaturing gel composed of urea and polyacrylamide (8.3 M urea, 8% acrylamide, and 0.2% bisacrylamide) was used for electrophoretic separation of RNA and subjected to northern blot in 1 × TBE buffer [50 mM Tris-borate (pH 8.3) and 1 mM EDTA]. The molecular-weight markers (low-range RNA ladder; Fermentas, Waltham, MA, USA) corresponding to the band in a lane was excised and stained with 5 mg/mL of ethidium bromide. It was then photographed under UV light beside a reference ruler. The remaining gel was electroblotted for 20 min in 1 × TBE buffer onto a Hybond-N membrane at 150 mA (15–25 V). Nucleic acids in the membranes were cross-linked by exposure to UV light for 5 min. Then 2 × SSC (1 × SSC contains 0.15 M NaCl and 15 mM sodium citrate) was used to wash all membranes (Sambrook and Russell, 2001). Northern hybridizations were performed according to recommended protocols (DIG filter hybridization; Roche) for using digoxigenin (DIG)-labeled DNA probes.
The β-galactosidase activity assay was performed as previously described36. The enzyme activity was normalized in Miller units of bacterial culture to the OD600 value. In order to induce the expression of strains containing pBBR1MCS-5 or pET-28a derivatives, 0.1 mM IPTG was added to cultures.
In view of the fact that LipA is an intracellular lipase, LipA activity was measured as the activity of whole-cell lipase. According to previously described methods [5], bacterial samples were prepared and 30 μL of p-nitrophenyl caprylate [pNPC; 2.9 mL 50 mM Tris–HCl (pH 9.0) and pNPC (10 mM pNPC in acetonitrile)] was used to determine lipase activity. The reaction mixture containing 70 μL of the cell sample was pre-heated for 5 min at 55 °C and centrifuged at 12,000 rpm for 2 min at 4 °C. The amount of pNP released in the supernatant was determined spectrophotometrically by measuring absorbance at 600 nm. One unit of enzyme activity (U) was defined as the amount required to release 1 μmol of p-nitrophenol/min. Lipase activity was expressed as U/mL*OD600.
This article does not contain any studies with human participants or animals performed by any of the authors.
