Industrial water samples from different geological areas of districts Kasur and Sheikhupura, Punjab, Pakistan were collected in sterilized polythene bags. All samples were brought to laboratory and different physico-chemical parameters were analyzed within 24–48 h. Bacterial load of the samples was determined by making serial dilutions of 10⁻¹ to 10⁻⁴ from 1% initial water sample. In order to isolate the individual colonies, 100 μl of the 10⁻³ and 100⁻⁴ dilutions were spread on LB agar plates containing different concentrations of HgCl₂ ranging from 1 to 20 μg/ml. The plates were incubated at 37°C for 24 h. The single isolated and HgCl₂ resistant colonies were selected and re-streaked on new LB agar plates without HgCl₂ to get purified colonies. All Hg-resistant and purified bacterial cultures were stored as glycerol stocks (30%) at−80°C.

Highly Hg-resistant isolate AZ-15 was biochemically characterized by different tests viz., Gram staining, shape, motility, spore formation, catalase, oxidase, oxygen requirement, MacConkey agar growth, indole production, methyl red, Voges-Proskauer, citrate (Simmon) agar growth, and H₂S production by following the protocols of Cappuccino and Sherman (2008). The bacterial genomic DNA of AZ-15 was extracted by genomic DNA purification kit. The universal primers were used for the amplification of 16S rRNA gene using BIOER XP-thermal cycler (Normand, 1995). The amplification conditions for both genes were set as: 5 min at 95°C, 35 cycles of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C, with a final 5 min chain elongation at 72°C. The sequence results obtained from Macrogen sequencing core facility, Korea were checked through nucleotide blast and submitted to NCBI GenBank.

The merF gene (wild type) of highly mercury resistant Enterobacter sp. AZ-15 was amplified using the primers given in Table 1. The amplified product of 246 bp was digested with the restriction enzymes Xhol and Sphl, and purified by gel extraction kit. The digested merF gene was ligated with the Xhol-Sphl-cleaved expression vector pET31b(+) (Kuliopulos et al., 1987). The genetic codons encoding cysteine residues in the native sequence of merF gene were mutated into codons encoding serine residues using site directed mutagenesis kit by designing two sets of primers (Table 1). Both sequences of merF gene (merF wild type and mutated merF) along with translated peptide sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov/) and obtained accession numbers as MF185183 and MF185184, respectively. The designed recombinant plasmid containing mutated merF (merFm) gene was transformed into DH5α competent cells and identified by using same restriction enzymes on agarose gel. The nucleotide sequence of recombinant pET31b(+) containing merFm gene was confirmed by DNA sequencing facility provided marketplace, University of California, San Diego (USA). Then the recombinant plasmid pET31b(+) was transformed into C43(DE3) competent E. coli cells. After transformation, stable C43(DE3): pET31b+merFm clones were screened and stored as glycerol stocks (20% of glycerol) at −80°C.

The expression of recombinant merFm gene was optimized in LB medium and unlabeled M9 medium supplemented with different concentrations (0.2, 0.5, and 1 mM) of isopropyl-β-D-thiogalactoside (IPTG) at different time intervals (2, 4, 6, 7 h, and overnight) of incubation. For expression of isotopically labeled MerFm protein, 5 ml of LB medium with final concentration of 50 mg/l of cabenicillin was inoculated with 5 μl of clone C43(DE3): pET31b+merFm glycerol stock. After 5 h incubation at 37°C, 1 ml of starter culture was used to inoculate 50 ml of M9 minimal medium (per liter) containing Na₂HPO₄ 7.0 g, KH₂PO₄3.0 g, NaCl 0.5g, CaCl₂ 0.1 mM, MgSO₄ 1 mM, thiamin 50 mg, d-glucose 10 g, and ¹⁵N-(NH₄)₂SO₄ 1 g containing 50 mg/l of carbenicillin. The culture was kept at 300 rpm at 37°C for overnight and 50 ml culture was poured into 450 ml of same ¹⁵N labeled-M9 medium. The cells were allowed to grow at 37°C in shaking incubator until the optical density (O.D₆₀₀) of 0.6 was achieved. The expression of recombinant plasmid (KSI_MerFm_His-tag) was achieved by adding 1 mM of IPTG as a final concentration and kept on shaking for another 7 h on same conditions. The pellet of C43(DE3) cells containing recombinant protein was obtained by subsequent harvesting at 7,000 rpm for 30 min at 4°C.

The cell pellets were resuspended in 30 ml lysis buffer (50 mM Tris hydrochloride, 15% glycerol (v/v), 1 mM NaN₃, pH 8.0). After 10 min of incubation at room temperature, cell lysate was sonicated by a probe sonicator (www.fishersci.com) for 5 min (cycles: 5 s on/10 s off) on ice and then centrifuged at 17,000 rpm for 30 min at 4°C. The pellet at this stage contains purified inclusion bodies containing more than 90% of MerFm fusion protein. The pellet containing inclusion bodies was then resuspended in the nickel column binding buffer (20 mM Tris hydrochloride, 500 mM NaCl, 6 M guanidinium hydrochloride (GndCl), 5 mM imidazole, pH 8.0) by tip sonication, and centrifuged again at 19,000 rpm for 1 h to remove remaining protein, lipids, and lipid-associated debris. The supernatant was loaded onto a Ni-NTA His-Bind Resin column, pre-equilibrated with binding buffer. The column was then washed with washing buffer (20 mM Tris hydrochloride, 500 mM NaCl, 6 M guanidinium hydrochloride (GndCl), 50 mM imidazole, pH 8.0). The fusion protein was eluted with elution buffer (20 mM Tris hydrochloride, 500 mM NaCl, 6 M GndCl, 500 mM imidazole, pH 8.0) and concentrated to 30 ml using an Amicon stirred concentrator cell with YM10 filter membrane and dialyzed against ddH₂O in a 10,000 kDa MWCO dialysis membrane with four water changes until the protein precipitates out of solution. The precipitated protein was then lyophilized.

The lyophilized protein was dissolved in 70% formic acid solution and cleaved by addition of a three-fold excess of cyanogen bromide (CNBr) for 3–5 h in the absence of light. The cleavage reaction was stopped by adding double volume of 1M NaOH. The cleaved polypeptide was dialyzed by using 3,500 kDa MWCO dialysis membrane against water and then lyophilized.

Cleaved protein was re-dissolved in 4 ml phosphate SDS buffer (100 mM Na₂HPO₄, 4 mM SDS, I mM EDTA, lmM NaN₃, pH 8.2) and 1 ml 1 N NaOH. Size-Exclusion gel chromatography using a Sephacryl S-200 column equilibrated with phosphate-SDS buffer on a Pharmacia FPLC system used to separate the KSI fusion partner from the pure MerFm protein. Fractions containing MerFm were pooled and concentrated down to 30 ml using an Amicon Stirred cell with a 3,500 MWCO membrane. The pure protein was then dialyzed against ddH₂O (with 40 mM β-mercaptoethanol for native MerFm) to remove SDS for six times with time interval of 12 h until the protein precipitates. Each time the protein was centrifuged out of solution, quickly lyophilized, and stored at −20°C for further use, Yields of MerFm were approximately 2–3 mg/l of cell culture. SDS-PAGE was run to monitor the purification, with the final native MerFm protein.

All NMR samples were prepared by resolubilizing the lyophilized protein in NMR buffer (10 mM Na₂HPO₄, 500 mM d₂₅-SDS, lmM NaN3, pH 6.0). The protein was then incubated at 40°C for 30 min with intermittent bath sonication and centrifuged at 13,000 rpm for I5 min to remove undissolved protein and other debris. The labeled peptide samples were prepared in different concentrations 10, 40, and 75% of D₂O. Two-dimensional heteronuclear single quantum coherence (HSQC) spectra of uniformly ¹⁵N-labeled MerFm were recorded on Bruker DMX 600 spectrometer equipped with a triple resonance probe, three-axis pulsed field gradients, a deuterium lock channel and operated at a ¹H NMR frequency of 600 MHz with total recording time 7 h. The other parameters for obtaining ¹⁵N–¹H fast Heteronuclear Single Quantum Cohererance (fHSQC) spectra include pulse sequence with 1,024 points in t2 and 256 points in t1 and 50°C using a 1.5 s recycle delay. The spectra were processed by using Sparky 3 [Goddard and Kneller, University of California San Francisco (UCSF), USA].

An experiment was designed to check the detoxification potential of Enterobacter sp. at a lab scale. Mercury resistant (Hg^R) bacterial strain AZ-15 and mercury sensitive strain (Hg^S) Enterobacter cloacae ZA-15 (KJ728671) with equal number of cells (cfu/ml) were inoculated separately in flasks containing 30 mL of LB medium, supplemented each flask with 20 μl/ml of HgCl₂ to determine the detoxification efficiency of mercury. The flasks were incubated at 37°C for up to 8 h with 2 h of time intervals at 120 rpm of agitation in triplicates. After incubation, cultures were spun down at 14,000 rpm for 15 min and detoxification of Hg²⁺ was estimated by dithizone method (Elly, 1973; Khan et al., 2006). For scanning electron microscopy (SEM), one drop of 48 h culture was diluted into 15 ml of ddH₂O and dried on carbon surface slide. The topographical, morphological and crystallographic information of bacterial cell membrane was monitored using Scanning Electron Microscope (SEM) (JEOL-JSM-6480).

The phylogenetic analysis of Enterobacter sp. AZ-15 with already reported bacteria on the basis of 16S rRNA gene was done by Mega 6.0 software (Tamura et al., 2013). The results of lab scale mercury detoxification experiment were subjected to mean, standard deviation, analysis of variance (ANOVA) by using SPSS V.20 software (IBM, 2011). The hydropathy plot predicting membrane spanning regions was built by SWISS-MODEL homology (www.expasy.org).

Industrial water samples collected from different geological sites (tanneries) of district Kasur and Itehad chemicals limited, district Sheikhupura, Lahore were checked for physico-chemical properties such as temperature, pH, Hg²⁺ concentration, bacterial load in the presence of metal stress of HgCl₂. Data indicate the level of pH in water sample falls in the range of 6.47–7.65. All samples (100%) were within the pH ranges of WHO drinking water guidelines (WHO, 2004) but the value of HgCl₂ which range from 5to 17 μg/ml has crossed limit described by WHO (1 μg/ml) (Javendra, 1995). The bacterial count in these samples is also variable, ranging from 2.1 × 10² to 2.9 × 10⁵ as presented in Table 2. A total 30 bacterial strains including Gram positive and Gram negative were screened on the basis of showing resistance against different concentrations of HgCl₂.

The most highly Hg-resistant bacterial isolate AZ-15 showing growth at 20 μg/ml of HgCl₂, was characterized on the basis of colony morphology and biochemical tests. The results are shown in Table 3. Mercury resistant bacterial isolate was identified by 16S rDNA sequencing (≈1.5 kb) as Enterobacter sp. with accession number KU558920. Other close matches to Enterobacter sp. AZ-15 (KU558920) includes E. cloacae strain E717 (EF059865), Enterobacter sp. BSRA3 (FJ868807), E. cloacae strain NBRC 13535 (NR113615), E. cloacae strain DSM 30054 (NR117679), E. cloacae strain RPR-CCFL3 (KR611993) and Enterobacter sp. Wy2-D9 (JN986806). Enterobacter sp. GJ1-11 (EU139848), Enterobacter sp. BAB-3140 (KF984440), and Enterobacter sp. STJ12 (KC833508) represent as an out group in this phylogenetic analysis (Supplementary Figure 1).

As shown in Figure 1A, the gene construct merFm was designed by ligation of wild type merF gene in pET31b(+) vector. The deduced amino acid sequence of merF gene showed two cysteine residues at the positions 71 and 72. The cysteine residues were mutated into serine residues in merFm gene by site-directed mutagenesis which results no cysteine residue in the final merFm gene construct as shown in Figure 1B. The salient features of construct include T7 promoter, ketosteroid isomerase (KSI) fusion partner, 5′ overhanging sequences compatible with SphI and XhoI restriction enzyme sites, within restriction sites; a transcription initiation codon (ATG) of merFm followed by encoding region of respective protein and a termination codon and a region encoding a hexahistidine amino acid tag and T7 terminator. His-tag sequence is for ease of purification and the KSI peptide sequence is designed to form inclusion bodies. After the transformation of recombinant plasmid into competent DH5α E. coli cells, the plasmid DNA was purified at concentration of 45 mg/ml. The plasmid was then transformed into E. coli C43(DE3) cells for expressing high concentration of MerFm recombinant protein and is thus protected from proteolysis. The fusion protein is non-toxic to the E. coli host cell, and is expressed at levels up to 20% of total cellular protein in E. coli strain C43(DE3). This approach to the production of MerFm in E. coli may be generally applicable to other membrane proteins. We have used it successfully with several other small membrane proteins.

For optimization of recombinant protein MerFm, three different concentrations, 0.2, 0.5, and 1 mM of IPTG were used at different post-induction time (h) viz., 2, 4, 6, 7, and overnight. The high expression of recombinant protein MerFm was achieved with 1 mM of IPTG and 7 h of post-induction time as shown in lane 12 of Supplementary Figure 2. The same conditions were maintained for expression of ¹⁵N-labeled MerFm fusion protein. After expression, the separation and purification of ¹⁵N-MerFm were performed by following the procedure described in detail in the methodology section. Briefly, the recombinant/fusion protein formed as inclusion bodies was separated from the total cell lysate by centrifugation. The fusion protein was partially purified by nickel affinity chromatography. The data in Supplementary Figure 3, illustrate the expression and isolation of inclusion bodies of full-length MerFm by Ni-NTA column. Lane 1 is showing Mark12™ unstained protein ladder (www.lamdabio.com). After the post-induction period of 7 h, the cells were lysed by sonication and centrifuged. After centrifugation, the supernatant was discarded which contained the total cellular proteins except inclusion bodies is shown in lane 2 of a 12% SDS-PAGE. Lane 3 contains the inclusion bodies solubilized in binding buffer and lanes 4 and 5 show the soluble fraction (flow through) and washing of other proteins with washing buffer, respectively except the targeted fusion protein. The lanes 6–10 show that the insoluble fractions (inclusion bodies) containing primarily the targeted fusion protein. After elution from the nickel column, the KSI fusion partner was cleaved from the MerFm polypeptide in the presence of CNBr at methionine for obtaining purified native MerFm. The lane 2 in Figure 2A shows the cleavage of fusion protein and the lane 3 contains the little bit impurity of KSI fusion partner while lanes 4–8 show the purified fractions ¹⁵N-MerFm after size-exclusion chromatography (FPLC). Figure 2B shows the peaks of ¹⁵N-MerFm and KSI in FPLC.

The ¹H-¹⁵N HSQC spectra of MerFm were obtained with the three concentrations of D₂O as shown in Figure 3. The high quality spectrum of 15N-MerfM was obtained with 40% concentration of D₂0 (Figure 3B). 1H-15N HSQC spectra were recorded under standard solution NMR conditions to confirm the purity and correct folding of the MerFm protein. The two-dimensional HSQC spectrum of ¹⁵N enriched MerFm contains the correct number of crosspeaks/resonances for each amide in the protein corresponding to side chain and tryptophan amide groups. This confirms that no modification of the tryptophan residue occurred during CNBr cleavage. Notably, the crosspeaks of six glycines in full-length MerFm are also present in the spectrum (Figure 3B). This is prerequisite for the preparation of well-behaved protein in phospholipid bilayer samples for solid-state NMR studies.

In lab scale experiment, the detoxification of Hg²⁺ by selected bacterial strain indicated 5 (25%), 10 (50%), 18 (90%), and 18 (90%) μg/ml detoxification of Hg²⁺ out of 20 μg/ml (100%) at 2, 4, 6 and 8 h of incubation, respectively by mercury resistant Enterobacter sp. AZ-15 (p < 0.05). The mercury sensitive strain E. cloacae ZA-15 was used as a negative control (Figure 4A). In SEM analysis, the accumulation of Hg was seen on the cell surface after 8 h of incubation with 20 μg/ml in LB medium. The SEM image showed crystalline structures of Hg deposited on the surface of cell membrane while in negative control, no crystalline structure was observed (Figure 4B). From this experiment, it is evident that Enterobacter sp. AZ-15 can accumulate Hg which confirms its detoxification potential.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.