Media and growth conditions
Aerobic cultures were grown in either LB (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl) or a modified version of the original M1 medium [9] with 30mM lactate as the electron donor. The modified M1 medium used in this study contains buffer/salts (3mM PIPES buffer, pH 7.0, 28mM NH4Cl, 1.34mM KCl, 4.4mM NaH2PO4, 125mM NaCl), vitamins [81.8nM D-biotin (vitamin B7), 45.3nM folic acid (vitamin B9), 486.4nM pyridoxine HCl (vitamin B6), 132.8nM riboflavin (vitamin B2), 133.6nM thiamine HCl (vitamin B1), 406.2nM nicotinic acid (vitamin B3), 209.8nM D-pantothenic acid, 0.74nM vitamin B12, 364.6nM p-aminobenzoic acid, 242.4nM lipoic acid], minerals [78.5μM nitriloacetic acid (trisodium salt), 249.1μM MgSO4 · 7 H2O, 29.6μM MnSO4 · 1 H2O, 171.1μM NaCl, 3.6μM FeSO4 · 7 H2O, 6.8μM CaCl2 · 2 H2O, 4.2μM CoCl2 · 6 H2O, 9.54μM ZnCl2, 0.4μM CuSO4 · 5 H2O, 0.21μM AlK(SO4)2 · 12 H2O, 1.61μM H3BO3, 1.24μM Na2MoO4 · 2 H2O, 1.01μM NiCl2 · 6 H2O, 0.76μM Na2WO4 · 2 H2O], and amino acids (135.9μM L-glutamic acid, 114.8μM L-arginine, 190.3μM DL-serine). Anaerobic cultures were grown in modified M1 medium with 30mM lactate as the electron donor and 30mM sodium fumarate as the electron acceptor. Anaerobic conditions in broth cultures were achieved by treating cultures in sealed test tubes using Oxyrase for Broth (Oxyrase, Inc., Mansfield, Ohio) as per the manufacturer’s instructions.
All S. oneidensis cultures were grown at 30°C, while E. coli cultures were grown at 37°C. Cultures containing both E. coli and S. oneidensis were grown at 30°C. Antibiotics were used at the following concentrations: Gentamicin (Gm): 5 μg/ml; Tetracycline (Tc): 10 μg/ml for E. coli; 1 μg/ml for S. oneidensis, [we used a lower concentration of Tc for selection of S. oneidensis than for E. coli because we found that the minimum inhibitory concentration (MIC) of Tc for S. oneidensis MR-1 is <1 μg/ml (data not shown)]; Kanamycin (Km): 25 μg/ml; Ampicillin (Amp): 100 μg/ml.
For growth curves, 5ml LB Km cultures of S. oneidensis strains were inoculated from frozen permanent stocks and aerobically outgrown overnight (10–12 hours). The overnight cultures were diluted in LB Km to an ABS600 ≅ 0.1 or in modified M1 Km to an ABS600 ≅ 0.025 and aerobically outgrown to log phase (ABS600 ≅ 0.4-0.8). These exponentially growing cultures were then diluted to an ABS600 ≅ 0.1 (LB Km) or to an ABS600 ≅ 0.025 (modified M1 Km). Aerobic cultures (15-20ml) were grown in 125mL Erlenmeyer flasks shaken at 250RPM. Anaerobic cultures (15ml) were grown in sealed test tubes without shaking. Culture densities (ABS600) were monitored spectrophotometrically, and culture titers (CFU/ml) were determined by plating serial dilutions of cultures on LB Km plates.
Construction of the S. oneidensis hfq∆ mutant and hfqrescue construct
To generate a null allele of hfq (So_0603 [12]) we deleted most of the hfq open reading frame and replaced it with a promoterless lacZ/gentamicin resistance gene cassette from pAB2001 [13]. We first PCR amplified a 5′ fragment using the primers GGCCCCGGGTAGAGCAAGGCTTTATTGATGAGGTAGC and GGCGCATGCGTCTTGTAAAGATTGCCCCTTAGCC and a 3’ fragment using the primers GGCGCATGCACGATATGCCAAGTGGCGAATAAGG and GGCGGTACCAGCTCGTTGGGCGAAAATATCCAAAATCAG. Following restriction (restriction endonucleases purchased from New England Biolabs, Ipswich, MA) of the 5′ PCR fragment with XmaI and SphI and restriction of the 3’ PCR fragment with SphI and KpnI, the two fragments were simultaneously ligated into pBSKS II + [14] that had been restricted with XmaI and KpnI. A 4.5kb SphI fragment from pAB2001 was then inserted into the SphI site of this plasmid to generate pBS-hfq∆. The XmaI-KpnI fragment from pBS-hfq∆, which contained the lacZ/gentamicin-disrupted hfq gene, was then cloned into XmaI/KpnI restricted pDMS197 [15], a R6K ori plasmid. The resulting plasmid, pDMS197-hfq∆ was transformed into E. coli SM10λpir [16], mated into S. oneidensis MR-1 [9], and Gmr/Tcr single crossover recombinants were isolated. Following growth in LB liquid without selection, cultures of these single crossovers were plated to LB plates containing Gm, 5% sucrose (w/v), and 0.1% NaCl (instead of omiting NaCl to increase the likelihood of isolating an hfq mutant in the event that loss of hfq resulted in cells sensitive to hypoosmotic conditions). Gmr Sucr Tcshfq∆ mutant candidates were screened via PCR and DNA sequencing of the disrupted region. The sequence of the primers used for diagnostic PCR in Figure 1 are as follows: A (hfq 5’ diagnostic) - ATAATGTGGTGCAATTTGCC; B (lacZ 5’ out) - CGTTGTAAAACGACGGGATCG; C (aacC1 3’out) - GATGCACTTTGATATCGACCC; D (hfq 3’ diagnostic) - GAGTCCAACCACGCACTAGG.
To generate an hfq rescue construct, we PCR amplified a 1.3kb genomic fragment containing the S. oneidensis MR-1 hfq coding sequence and ~1kb upstream of the hfq open reading frame. Based on hfq promoter analysis in E. coli, this fragment likely contains the native promoters for S. oneidensis hfq [17]. A PCR product was generated using the 5’ primer GGCAAGCTTCAGGAAAAACGGCTTTAGCTCTCG and the 3’ primer GGCGGTACCACTAAACCTTATTCGCCACTTGGC. Following restriction with HindIII and KpnI, this PCR product was ligated to HindIII/KpnI restricted pBBR-1MCS2 [18]. The resulting plasmid, pBBR1-hfq, was transformed into E. coli S17-1λpir [19] and mated into S. oneidensis strains. In our hands, the pBBR1-MCS2 based vectors were stably maintained in S. oneidensis strains after 30 hours in LB Km cultures and after 120 hours in modified M1 Km cultures (data not shown).
Western blot analyses
3ml aliquots of S. oneidensis cultures were pelleted in a microcentrifuge for 2’ at 20300 x g. Bacterial pellets were frozen at −80°C, thawed, and then treated with Bacterial Protein Extraction Reagent (B-PER) in the presence of 100μg/ml lysozyme, 5U/ml DNAse I, and 1X Halt Protease Inhibitor Cocktail. Protein was quantified using the Pierce BCA Protein Assay Kit as per manufacturers instructions (all reagents were obtained from Thermo Scientific, Rockford, IL).
For western blot analysis, 90μg of protein per lane was size fractionated at 4°C using Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA). Proteins were then transferred to an Immobilon-PSQ PVDF membrane (EMD Millipore, Billerica, MA). Equivalent protein in different lanes was verified by Ponceau S staining of the membrane (data not shown). The membrane was blocked for 1 hour at room temperature using LI-COR Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) and probed with a 1:5000 dilution of primary antibody, rabbit anti-E. coli Hfq [20] overnight at 4°C. The blot was washed 4 times for 5 minutes each with PBS-T and then probed with a 1:10000 dilution of goat anti-rabbit secondary antibody conjugated to IRDye 800CW Infrared Dye (LI-COR Biosciences, Lincoln, NE) for 45 minutes at room temperature (~22°C). The blot was washed with PBS-T 4 times for 5 minutes each and then rinsed with PBS to remove residual Tween 20. The blot was then imaged on a LI-COR Odyssey infrared scanner. Protein in Figure 1C was harvested from 24 hour old LB Km cultures. Older cultures consistently accumulated higher levels of Hfq protein, though our western blot results were consistent regardless of culture age at harvest; we never observed Hfq protein in the hfq∆/empty vector cultures (Figure 1C and data not shown).
Chromium reduction assays
Chromium reduction assays were performed using a diphenylcarbazide-based quantitative, valence state specific, colorimetric assay for Cr(VI) [21]. Log phase cultures (ABS600 ≅ 0.5-0.8) grown in modified M1 medium were diluted to ABS600 ≅ 0.4 in modified M1 medium that had been prewarmed to 30°C. The cultures were transferred to sealed test tubes and treated for 30 minutes at 30°C with Oxyrase for Broth (Oxyrase, Inc., Mansfield, Ohio) to remove oxygen. Following addition of 100μM K2CrO4, cultures were incubated without shaking in a 30°C water bath in sealed test tubes. 1ml aliquots of cultures were periodically removed and added to 13mm borosilicate glass tubes containing 0.25ml of a 0.5% diphenylcarbazide solution in acetone and 2.5ml 0.28N HCl. Following vortexing, ABS541 values for individual samples were measured in a SPECTRONIC 20D+ spectrophotometer (Thermo Scientific, Rockford, IL).
Oxidative stress assays
Overnight cultures grown in LB Km were diluted to an ABS600 ≅ 0.1. These cultures were outgrown for 2–3 hours to exponential phase (ABS600 ≅ 0.4-0.6) then diluted to an ABS600 ≅ 0.2. Following five minutes of aerobic growth, cultures were treated with H2O (mock), 0.4 mM H2O2 to induce peroxide stress, or 5 mM methyl-viologen (paraquat) to induce superoxide stress. Cultures were then grown aerobically for 15 minutes. Following treatment, each culture was serially diluted in triplicate in phosphate-buffered saline (PBS, pH 7.4). The dilutions were plated to LB Km plates within five minutes of harvest and grown overnight before scoring.