M. acetivorans strains and growth.
M. acetivorans strain WWM73 was the parent strain for all experiments. All strains of M. acetivorans are listed in Table 1. The strains were grown in anoxic HS medium at 35º C as previously described [37]. HS medium was prepared inside an anaerobic chamber (Coy Laboratories) containing 75% N2, 20% CO2, and 5% H2. The medium was supplemented with 2 µg/mL puromycin when required. Growth experiments were performed in Balch tubes containing 10 ml of HS medium reduced with 1.5 mM DTT. 125 mM methanol or 50 mM TMA or 100 mM acetate, 1 mM sulfide and/or 3 mM cysteine and 18 mM NH4Cl were added from anaerobic sterile stock solutions prior to inoculation where indicated. The optical density was measured at 600 nm using a spectrophotometer.
Construction of M. acetivorans sufCB CRISPRi repression strains.
All primers (Table S1) and gBlocks were designed using Geneious Prime software and purchased from Integrated DNA Technologies (IDT). For the construction of CRISPRi-dCas9 plasmids with a single gRNA, a gBlock containing either gRNA-suf1 or gRNA-suf2 (Table 3) was introduced into pDL734 separately as previously described [22]. Briefly, pDL366 and pDL367 (Table 2) were constructed by digesting pDL734 with AscI (New England Biolabs). The gBlock was then introduced into digested pDL734 using a Gibson Assembly Ultra Master mix kit (Codex DNA) as per the manufacturer’s instructions. E. coli WM4489 competent cells were transformed with Gibson Assembly reactions. Transformants were screened by PCR using primers P1 and P2. Next, M. acetivorans WWM73 was separately transformed with pDL366 and pDL367 using liposome-mediated transformation method [38]. Transformants were selected on anaerobic HS agar plates containing 125 mM methanol and 2 µg/mL puromycin. The plates were incubated at 35º C in an anoxic mason jar containing 2.5 mL of 2.5% w/v sulfide in a vial and incubated. Colonies were screened by PCR using two sets of primers P3/P4 and P5/P6 and positive colonies were selected and designated as DJL130 (pDL366) and DJL131 (pDL367). The strains were maintained in HS medium containing 125 mM methanol, 1 mM sulfide and 2 µg/mL puromycin.
For the construction of CRISPRi-dCas9 plasmid with two gRNAs (pDL371), pDL367 was digested with HpaI (NEB) and a gBlock containing gRNA-suf1 was introduced into HpaI-digested pDL367 using Gibson Assembly. Transformants were screened by PCR using primers P7 and P8. Next, M. acetivorans WWM73 was transformed with pDL371 as described above. Colonies were screened by PCR using two sets of primers P3/P4 and P5/P6 and positive colony was selected and designated as DJL133. The strain was maintained in HS medium containing 125 mM methanol, 1 mM sulfide and 2 µg/mL puromycin.
Gene expression analysis.
M. acetivorans cells were harvested at mid-log phase (OD600 of 0.4–0.5) by anaerobic centrifugation. Pellets were resuspended in 1 mL Trizol reagent (Ambion, Life Technologies) and stored at -80º C. RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research) followed by DNase treatment using the DNA-free DNA Removal kit (Invitrogen, Thermo Fisher Scientific). cDNA was synthesized from 300 ng of RNA using the iScript Select cDNA Synthesis kit (Bio-Rad). Gene expression analysis was done by qPCR using cDNA (300-fold dilution) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). Primers used for qPCR were designed using Geneious Prime software and purchased from IDT. sufB1 expression was analyzed using primers P41/42, sufC1 expression using primers P43/44, sufB2 expression using primers P45/46, and sufC2 expression using primers P47/48. Primers P39 and P40 were used to measure the expression of 16s rRNA (an internal control). The reactions were carried out in a CFX96 Real-Time PCR Detection system (Bio-Rad). The data were analyzed using ∆∆Cq calculation method.
Generation of M. acetivorans sufBC deletion mutant strains.
All deletion strains of M. acetivorans were generated using a CRISPR-Cas9 system with a few modifications [24]. All primers and gBlocks were designed using Geneious Prime software and purchased from IDT. Strain DJL63 was generated by introducing sufC2B2-editing DNA (homology repair template + gRNA1-suf2 + gRNA2-suf2) into pUC18 followed by introducing it to pDL238. Briefly, homology regions upstream and downstream of sufC2B2 were amplified by PCR using primers P9/10 and P11/12 respectively. Plasmid pDL248 was constructed by digesting pUC18 with BamHI-HF and introducing sufC2B2-editing DNA into digested pUC18 using Gibson Assembly. E. coli DH5α competent cells were transformed with Gibson Assembly reaction. Transformants were screened by PCR using P9 and P13. The sufC2B2-editing DNA was amplified by PCR using Q5 polymerase, primers P14/P15 and pDL248 as template. The amplified sufC2B2-editing DNA was introduced into AscI-digested pDL238 using Gibson Assembly followed by transformation into WM4489 competent cells. Transformants were screened by PCR to confirm the presence of plasmid pDL249 using primers P9 and P13. pDL249 was retrofitted with pAMG40 using Gateway BP Clonase II enzyme mix (Invitrogen) followed by transformation into WM4489 cells. Transformants were screened by PCR using the primer set P16/P17 and the plasmid was designated pDL250. Next, M. acetivorans WWM73 cells were transformed with pDL250. Transformants were selected on anaerobic HS agar plates containing 125 mM methanol and 2 µg/mL puromycin as described above. Colonies were screened by PCR using primers that anneal outside the homology region (P18 and P19) and gene-specific primers (P20/21 for sufB2 and P22/23 for sufC2). To construct a markerless deletion mutant, cells were plated on HS plates containing 125 mM methanol and 50 µg/mL 8-Aza-2,6-diaminopurine sulfate (8-ADP) as described [24]. The colonies cured of the plasmid were screened by PCR using primers P24 and P25.
For the construction of DJL143 (suf1 deletion mutant) and DJL142 (suf1/2 deletion mutant), homology regions upstream and downstream of sufC1B1 were amplified by PCR using primers P26/27 and P28/29 respectively. Plasmid pDL381 was constructed by digesting pUC18 with BamHI-HF and introducing sufB1C1-editing DNA (homology repair template + gRNA1-suf1) into digested pUC18 using Gibson Assembly. E. coli DH5α competent cells were transformed with Gibson Assembly reaction. Transformants were screened by PCR using primers P13 and P26. The sufC1B1-editing DNA was amplified by PCR using Q5 polymerase, primers P15/30 and pDL381 as template. The amplified sufC1B1-editing DNA was introduced into AscI-digested pDL238 using Gibson Assembly followed by transformation into WM4489 competent cells. Transformants were screened by PCR to confirm the presence of plasmid pDL385 using primers P15 and P26. To introduce the second gBlock containing gRNA2-suf2, pDL385 was digested with HpaI and gRNA2-suf2 was introduced into HpaI-digested pDL385 using Gibson Assembly followed by steps mentioned above to construct a retrofitted plasmid (pDL387). M. acetivorans WWM73 and DJL63 were separately transformed with plasmid pDL387 to generate suf1 (DJl143) and suf1/2 (DJL142) deletion mutants. Transformants were selected on anaerobic HS agar plates as described above. Colonies were screened by PCR using primers that anneal outside the homology region (P31 and P32) and gene-specific primers (P33/34 for sufB1 and P35/36 for sufC1). To construct a markerless deletion mutant, cells were plated on HS plates containing 125 mM methanol and 50 µg/mL 8-ADP (Biosynth). The colonies cured of the plasmid were screened by PCR using primers P24 and P25.
All plasmids transformed into M. acetivorans were verified by DNA sequencing (Plasmidsaurus Sequencing). Additionally, deletions were confirmed by sequencing PCR-amplified upstream and downstream regions around the deleted genes.
Determination of total Fe-S cluster content in M. acetivorans cell lysate.
The total Fe- S cluster within M. acetivorans strains was determined by measuring total acid-labile sulfur content using the methylene blue method as previously described [39]. M. acetivorans cells were harvested at mid-log phase (OD600 of 0.4–0.5) by anaerobic centrifugation (8,000 x g for 15 minutes at 4º C). The cell pellet was resuspended in buffer containing 50 mM Tris, 150 mM NaCl, 1 mM Benzamidine, and 1 mM Phenylmethylsulfonyl fluoride and stored at -80º C using anaerobic vials. Cell pellets were lysed anaerobically by sonication on ice using a sonicator (QSonica) followed by anaerobic centrifugation (14,000 x g for 10 minutes). The cell lysate was collected, and the total protein concentration was determined using Qubit protein assay kit (Molecular probes, Life Technologies). The assay was performed in sealed vials containing protein samples. 1% zinc acetate and 12% sodium hydroxide were added to the vials containing samples and incubated at room temperature for 2 hours. The reactions were terminated by the addition of 5 mM N,N-Dimethyl-p-phenylenediamine dihydrochloride in 5 M HCl and 0.23 M FeCl3 in 1.2 N HCl followed by incubation at room temperature for 2 hours. The vials were quickly vented, solutions were spun down at 16,000 x g and absorbances were read at 670 nm using a spectrophotometer.
Construction of a Strep-His-SufC2B2 expression strain of M. acetivorans.
PCR was used to amplify sufC2B2 from M. acetivorans genomic DNA using Q5 high-fidelity DNA polymerase (NEB) as per the manufacturer’s instructions. Primers were designed and purchased from IDT. Restriction site NcoI was added at the 5’ end of sufC2 and restriction site NruI at the 3’ end of sufB2 using primers P37 and P38. The PCR product and plasmid pJA6 [40] were digested with NcoI and NruI using standard digestion protocol (NEB). Digested PCR product and pJA6 were ligated using T4 DNA ligase as per the manufacturer’s instructions. E. coli DH5α competent cells (NEB) were transformed with the ligation reaction. Transformants were screened by PCR using primer set P37/38 and confirmed by DNA sequencing (Eurofins). The pJA6 plasmid containing the sufC2B2 gene sequence was designated as pDL365. M. acetivorans strain WWM73 was transformed with pDL365 using a liposome-mediated transformation protocol as previously described [38]. Transformants were selected on HS agar plates as described above. Transformants were screened by PCR for the presence of strep-sufC2B2, and a positive transformant designated as strain DJL66 and maintained in HS medium containing 125 mM methanol, 1 mM sulfide and 2 µg/mL puromycin.
Western blot analysis.
M. acetivorans cells were harvested at mid-log phase (OD600 of 0.4–0.5) by aerobic centrifugation (8,000 x g for 10 minutes at 4º C). The cell pellet was resuspended in buffer A containing 50 mM Tris, 150 mM NaCl, 1% Tween, 1 mM Benzamidine, 2 mM EDTA and sonicated 3 times on ice using a sonicator (QSonica) followed by aerobic centrifugation (8,000 x g for 10 minutes at 4º C). The cell lysate was collected, and the total protein concentration was determined using Bio-Rad protein assay [41]. Protein sample (15 µg) was resolved in a 12% SDS-PAGE gel and transferred to a nitrocellulose blotting membrane (Amersham Protran 0.1 µm NC; GE Healthcare Life Sciences). The membrane was blocked for 20 minutes in blocking buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween (TBST), 5% milk and incubated overnight with the α-strep antibody (Qiagen). The membrane was washed three times with goat anti-mouse IgG antibody (GenScript) for one hour followed by washing three times with TBST again. The membrane was developed with Western blotting substrate (Thermo Scientific) and scanned using a FlourChem 8900 imaging system (Alpha Innotech).
Purification and characterization of Strep-SufC2.
M. acetivorans cells were harvested at an OD600 of 0.8 by anaerobic centrifugation (8,000 x g for 10 minutes at 4º C) and the pellets were stored at -80ºC. For purification of recombinant protein, all the steps were carried out in an anaerobic chamber (Coy Laboratories) containing 95% N2 and 5% H2. The cells were thawed and resuspended in buffer NP (50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) containing 4 µg/mL DNase I and 1 mM Benzamidine. The cells were sonicated 3 times followed by centrifugation (8,000 x g for 10 minutes at 4º C). The cell lysate was loaded on a chromatography column containing 1 mL of Strep-Tactin superflow plus resin (Qiagen) pre-equilibrated with 12 mL of buffer NP. The column was washed with 5 ml buffer NP. Protein was eluted in 3 mL buffer NP containing 2.5 mM desthiobiotin (NPD) and stored at -80º C. The protein concentration was determined using the Bio-Rad protein assay [41]. The purified protein was analyzed by 12% SDS-PAGE gel stained with Coomassie blue solution followed by de-staining. The protein bands were excised, trypsin digested, and sent for mass spectrometry (The University of Arkansas Statewide Mass Spectrometry Facility). UV-visible absorption spectrum was recorded using a Cary 60 spectrophotometer (Agilent Technologies) under anaerobic conditions.