Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs. Nickel-nitrilotriacetic acid resin and plasmid kits were obtained from Qiagen. FAD, FMN, Riboflavin, NADH, deamino-NADH, NADPH, Q1 and menadione were purchased from Sigma Chemical Co. (U.S.A).
2.2. Bacterial strains and growth conditions
OF4 strain 811M was grown at 30°C in a semi-defined medium containing 0.1% yeast extract with mineral salts and buffered with 0.1 M Na2
at pH 10.5 [7
]. E. coli
) lacking three major Na+
antiporters was routinely grown in LBK medium consisting of 1% tryptone, 0.5% yeast extract, and 0.6% KCl, pH 7.5 with 50 µg/ml kanamycin (Km). E. coli
) without NADH dehydrogenase was grown in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.5) with 50 µg/ml kanamycin. E. coli
strain Top 10 was grown in LB medium at pH 7.5. For complementation growth experiments, transformants of E. coli
KNabc were grown in 2 ml of LBK with different concentrations of NaCl at 37°C, with 250 rpm shaking, for 16 hours. Arabinose (0.5%, w/v) was added in some experiments. Transformants of E. coli
ANN0222 were grown on LB medium or M9 minimal medium (1X M9 salts, 2X 10−3
, 0.4% mannitol) with different concentrations of arabinose for induction [8
]. The growth medium of plasmid transformants also contained 100 µg/ml ampicillin (Ap).
2.3. Cloning of the putative NADH dehydrogenase genes
Genomic DNA of Bacillus OF4 was extracted with Ultraclean Microbial DNA Isolation Kit (MO BIO Laboratories). Primers were designed on the unpublished ndh-2A and ndh-2B nucleotide sequences (Genbank accession no. EU030627 and EU030628, respectively). Primers used for cloning the ndh-2A with an added C-terminal His6-tag were 5’-ACCATGGAAGTGATTAGTTTGAAAAAG-3’ (NDH-2AF) and 5’-CAATGGGTTTTTCCCTTTTTTC-3’ (NDH-2AH6R); The primers for the untagged version of ndh-2A were NDH-2AF and 5’-TTACAATGGGTTTTTCCCTTTTT-3’ (NDH-2AR); Primers used for cloning the ndh-2B with the additional C-terminal His6-tag were 5’-GAGGAATAATAAATGACGATGAAATATGTAATTATC-3’ (NDH-2BF) and 5’-CCCTCTGCGCCTTGATGCC-3’ (NDH-2BH6R); primers for the untagged form of ndh-2B were NDH-2BF and 5’-TTACCCTCTGCGCCTTGATG-3’ (NDH-2BR). PCR reactions were carried out in a MJ mini thermal cycler (Bio-Rad) with a Taq DNA polymerase (TaKaRa, ExTaq hot start version). The resulting four PCR products were ligated with pBAD TOPO vector (Invitrogen) and then transformed into E. coli Top10 on selective LB-ampicillin plates (pH 7.5) at 37°C. The correct insert direction was identified by restriction analysis of the recombinant plasmids. These recombinant plasmids were designated as pBAD-NDH-2A-HM, pBAD-NDH-2A-NM, pBAD-NDH-2B and pBAD-NDH-2B-N respectively. The pBAD-NDH-2A-HM and pBAD-NDH-2A-NM were digested by NcoI and the resulting bands of about 5.3 Kb were self-ligated respectively. These new constructs were designated as pBAD-NDH-2A and pBAD-NDH-2A-N. The pBAD cloning for the His-tagged constructs results in an additional 28 amino acids, including the six histidine residues, added to the C-terminus of the two proteins. The non-His-tagged NDH-2A has 405 amino acids and an Mr of 44,600. The native NDH-2B has 440 amino acids and an Mr of 48,600. With the His-tags, the sizes of NDH-2A and NDH-2B are 47,700 and 51,700, respectively. Sequence analyses were conducted in the Mount Sinai School of Medicine DNA Core Facility.
2.4. Complementation assays in E. coli mutant strains
pBAD-LacZ (pBAD-TOPO/lacZ/V5-His vector, Invitrogen), pBAD-NDH-2A, pBAD-NDH-2B and pBAD-OF4-Mrp were transformed into E. coli KNabc and E. coli ANN0222. The pBAD-OF4-Mrp from Bacillus pseudofirmus OF4, provided by Dr. Masahiro Ito (Toyo University), served as a positive control for E. coli KNabc complementation and was tested for the ability to complement E. coli ANN0222. The plasmid pBAD-lacZ was used as a negative control for both strains. For complementation of E. coli KNabc transformants, 10 µl of overnight pre-culture grown in LBK was inoculated into 2 ml of LBK medium with different concentrations of NaCl, and after 16 hours, the A600 was recorded. In some experiments, 0.5% of arabinose (w/v) was added. For complementation of E. coli ANN0222, transformants were grown on M9 minimal medium, with different concentrations of arabinose to vary the extent of induction and after 48 hours, the A600 was recorded. As a control, the E. coli ANN0222 transformants were grown on LB medium with different concentrations of arabinose to induce and after 16 hours, the A600 was recorded. All complementation assays were performed in duplicate in at least two separate experiments.
2.5. Isolation of everted membrane vesicles and assay of Na+/H+ antiporter activity
Liquid cultures were inoculated with 1% (v/v) of overnight pre-cultures of E. coli
KNabc transformants. The pre-culture and final cultures were grown in LBK at 37° C. When the cultures reached an A600
of 0.5, arabinose was added to the final concentration of 0.5% (w/v) except for the E. coli
KNabc/pBAD-OF4-Mrp, in which 0.005% arabinose was used. Cells were harvested at an A600
of 2.5 to 3.0 after induction. Everted membrane vesicles were prepared from E. coli
KNabc transformants as described [9
]. The buffers used in the preparation were 10 mM Tris-HCl, pH 7.5, containing 140 mM choline chloride, 0.5 mM dithiothreitol, 10% glycerol, a protease inhibitor tablet (Roche), 1 mM phenylmethylsulfonylfluoride (PMSF) and a trace amount of DNase I (Roche). Protein content was measured by the Lowry method using bovine serum albumin as the standard [10
]. Assays of monovalent cation/H+
antiport were conducted using acridine orange (AO) as a fluorescent probe of the transmembrane pH gradient (ΔpH, acid in) as described [11
]. The assay mixtures were made up to a total volume of 2 ml containing: 10 mM Tris-HCl (pH from 6.5 to 9), 140 mM choline chloride, 5 mM MgCl2
, 1 µM AO and 75 µg of vesicle protein. Respiration was initiated by the addition of Tris-succinate to a final concentration of 2.5 mM. Fluorescence was monitored with a Shimadzu RF-5301PC fluorescence spectrofluorophotometer at excitation and emission wavelengths of 420 nm and 500 nm, respectively. All assays were conducted in duplicate or triplicate in 2–3 independent experiments on different preparations.
2.6. Expression and Purification of His-tagged NDH-2A and NDH-2B
An overnight culture of E. coli Top10/ pBAD-NDH-2A was inoculated (1%) into LB medium, with 225 rpm shaking at 30° C. At an A600 of 0.8, 0.002% arabinose was added. The cells were harvested after 3 hours induction, washed twice with a solution containing 50 mM Tris-HCl, pH 8, and stored at −80° C. For the expression of NDH-2B, the E. coli Top10/pBAD-NDH-2B was cultured at 37° C with 225 rpm shaking. At an A600 of 0.5, 0.002% arabinose was added. The cells were harvested after 5 hours induction, washed twice with a solution containing 50 mM Tris-HCl, pH 8, and stored at −80° C.
After thawing, the cells were suspended in lysis buffer containing 50 mM Tris-HCl, pH 8, 300 mM NaCl, 10 mM imidazole, a protease inhibitor tablet (Roche), 1 mM PMSF and a trace amount of DNase I (Roche). The cells were broken in a French Press Cell under 10,000 p.s.i. pressure. The broken cell suspensions were centrifuged at 14,000 g for 15 min to precipitate unbroken cells and debris. The membrane vesicles were pelleted by ultracentrifugation at 250,000 g (Beckman Ti60 rotor) for 1 hour at 4° C and the resulting supernatant was subsequently used for purification. All subsequent steps of purification were performed at 4° C. His-tagged protein was purified by chromatography using Ni-NTA resin (Qiagen). The resin was pre-equilibrated with lysis buffer containing 50 mM Tris-HCl, pH 8, 300 mM NaCl and 10 mM imidazole. Two ml of 50% Ni-NTA slurry was added to the lysate and then gently mixed for 1 hour at 4°C. The lysate-Ni-NTA mixture was transferred to a small column and washed twice with 6 ml wash buffer containing 50 mM Tris-HCl, pH 8, 300 mM NaCl, 20 mM imidazole. The bound enzyme was eluted with elution buffer containing 50 mM Tris-HCl, pH 8, 300 mM NaCl, 250 mM imidazole and the active, yellow fractions were pooled. Glycerol was added to 30% (w/v), and the enzyme was quick-frozen in liquid nitrogen and stored at −80° C. In some cases, during the purification of His6-tagged NDH-2A protein, 20 µm FAD was added to all the buffers as detailed in the Results section. The NDH-2B was concentrated using Amicon Ultra-15 (Millipore) with a pore size of 10 KDa before the storage. It was found that concentration of NDH-2A resulted in loss of flavin, so the Ni-NTA pooled peak fraction was not concentrated before freezing.
2.7. Flavin analysis: thin layer chromatography, UV/visible spectra, Fluorescence spectroscopy
For the spectral analysis and thin layer chromatography, a larger scale preparation of NDH-2A was isolated without added FAD using a minimal volume of Ni-NTA resin so that it eluted in a concentrated form. It was then immediately used for the spectral analysis, due to the lability of its flavin moiety. As a consequence, the spectra of NDH-2A were recorded on samples in the Ni-NTA elution buffer, i.e., 50 mM Tris-HCl, pH 8, 300 mM NaCl, and 250 mM imidazole, at a concentration of 13.5 mg/ml. The spectra of NDH-2B, which was 2.65 mg/ml, were recorded from samples in 50 mM Tris-HCl pH 8.0 buffer that also contained 30% glycerol. UV-visible spectra of the purified enzyme were recorded at room temperature in a final volume of 0.5 ml with a Shimadzu UV-2501PC UV-vis recording spectrophotometer. Fluorescence spectra of the purified enzyme were also recorded at room temperature with a Shimadzu RF-5301PC fluorescence spectrofluorophotometer with a slit width of 5 nm for both excitation and emission wavelengths (2 ml volume). Fluorescence spectra of NDH-2A were obtained by excitation at 477 nm and monitoring of emission at 522 nm. Fluorescence spectra of NDH-2B were obtained by excitation at 475 nm and emission at 520 nm.
The type of flavin was determined by thin layer chromatography (TLC). Two different methods were used to extract the flavin from the purified proteins. For one method, the protein (about 5 mg) was boiled for 5 min followed by centrifugation which resulted in a yellow supernatant and an uncolored pellet. For the second method, the protein was precipitated with 10% trichloroacetic acid (TCA) for 30 min on ice followed by centrifugation. The TCA was removed from the yellow supernatant by washing 3 times with 2 volumes of ether [12
]. The supernatant samples were concentrated 15-fold under vacuum, and then were loaded onto silica gel plates (250 µm layer, 20×20 cm, catalogue no.4410222, Whatman, England). The plates were eluted in either solvent A (2% [w/v] Na2
in water) or solvent B (n-butanol/glacial acetic acid/water, 2:1:1), and flavins were detected as fluorescent spots upon ultraviolet irradiation.
2.8. Enzyme assays
All enzyme assays were performed at room temperature in a Shimadzu UV-2501PC UV-vis recording spectrophotometer. The assay volume was 1 ml and was buffered with either 50 mM BTP (bis-[tris(hydroxymethyl)methylamino]-propane) from pH 6–9.5 or citric acid-phosphate buffer from pH 3–7. The reductant and enzyme were added to different sides of the cuvette above the liquid and the reaction was initiated by rapid manual mixing. Between 1 and 10 µg of protein was used in the assays. In some assays, selected cations were added to determine if they affected activity. FAD (20 µM) was added in assays of NDH-2A. With the exception of ferricyanide oxidoreductase activity measurements, reactions were followed by the decrease in A340
, reflecting the oxidation of the electron donor (NAD(P)H or deamino-NADH, which were used at 200 µM). With each set of assays, the background activity in the absence of protein was determined and the background was subtracted from the activity observed in the presence of protein with oxygen as the electron acceptor [13
]. The corrected oxidase activity was then subtracted from the rates observed with enzyme and menadione or Q1 (50 µM) as the electron acceptor. For the electron acceptor ferricyanide, the reactions were monitored at A420
, using 1mM NAD(P)H or deamino-NADH and 1 mM K3
. For this set of reactions, the background activity without protein was subtracted from the enzymatic rates to yield the true enzyme activities. The following extinction coefficients (mM−1
) were used in the calculations: NADH (O2
or menadione as electron acceptor), 6.2 at 340 nm; NADH (Q1 as electron acceptor) [14
], 6.81 at 340 nm; ferricyanide, 1.0 at 420 nm [15
]. However, since we are defining one unit of enzyme activity as 1 µmole of NADH oxidized per minute, we have divided the ferricyanide reductase activity by 2 to reflect the activity on the basis of 2 electrons, i.e., per molecule of NADH. All assays were conducted in duplicate or triplicate in 2–3 independent experiments on different preparations.
2.9 Other biochemical analyses
Protein content was determined by the method of Lowry et al. [10
], using bovine serum albumin as a standard. Proteins were resolved on 12% SDS PAGE gels [16
]. The gels were stained with colloidal Coomassie Brilliant Blue G (National Diagnostics) or transferred to nitrocellulose membranes, and His-tagged proteins were detected by chemiluminescence (Pierce) with the INDIA-anti-His probe (Pierce).