Isolation and Characterization of Mercuric Reductase by Newly Isolated Halophilic Bacterium, Bacillus Firmus MN8
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Global J. Environ. Sci. Manage., 3(4): 427-436, Autumn 2017 DOI: 10.22034/gjesm.2017.03.04.008 ORIGINAL RESEARCH PAPER Isolation and characterization of mercuric reductase by newly isolated halophilic bacterium, Bacillus firmus MN8 M. Noroozi 1, M.A. Amoozegar 2, A.A. Pourbabaee 3,*, N.S. Naghavi 4, Z. Nourmohammadi 1 1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran 3Soil Science Department, Faculty of Agricultural Engineering and Technology, University College of Agriculture and Natural Resources, University of Tehran, Tehran, Iran 4Department of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Received 15 March 2017; revised 25 July 2017; accepted 7 August 2017; available online 1 September 2017 ABSTRACT: The current study was aimed at isolating and identifying the halophilic and halotolerant bacteria which can produce mercuric reductase in Gavkhuni wetland in Iran. Moreover, tracking and sequencing merA gene and kinetic properties of mercuric reductase in the selected strain were performed in this study. Soil samples were taken from Gavkhuni wetland and cultured in nutrient agar medium with 5% NaCl. To examine the tolerance of purified colonies to mercury, agar dilution method was administered. Similarly, the phylogenetic analysis based on 16SrRNA gene sequencing was conducted. To investigate enzyme activity of kinetic parameters, a spectrophotometer was used to measure the NADPH oxidation decrease at 340 n.m. The results showed that among the 21 halophilic and halotolerant strains isolated from Gavkhuni wetland, 4 were resistant to mercuric chloride. A strain designated MN8 was selected for further studies because it showed the highest resistance to mercury. According to phylogenetic sequencing of 16S rRNA gene and phenotypic characteristics, the strain was categorized in the Bacillus genus and nearly related to Bacillus firmus. This strain had merA gene. The mercuric reductase showed Vmax and Km values of 0.106 U/mg and 24.051 µM, respectively. Evaluation of different concentrations of NaCl at 37°C and pH=7.5 in mercuric reductase enzyme activity indicated that the enzyme shows 50% activity in concentration of 1.5 M. Optimum pH and temperature of enzyme activity were 7.5 and 35 °C, respectively. The results suggested that MN8 strain could be a proper candidate for bioremediation of mercury-contaminated environments such as industrial wastewaters. KEYWORDS: Halophilic bacteria; merA gene; Mercuric reductase enzyme; Mercury; Polymerase chain reaction (PCR). INTRODUCTION One of the most important environmental concerns environmental systems in small amounts and in is the contamination due to industrial heavy metals contaminated environments in high doses because of that are globally considered as risk factors with human activities. A few archaea and many halotolerant various impacts on human health and the environment and moderately halophilic bacteria are able to (Xiong et al., 2008; Giovanella et al., 2016). There tolerate metals through some mechanisms including are naturally heavy metals in the hypersaline enzymatic detoxification, metal efflux via specific transporters and extracellular metal sequestration *Corresponding Author Email: [email protected] by biopolymers (Voica et al., 2016). Exposure to Tel.: +98 26 3223 1787 Fax: +98 26 3223 1787 Note: Discussion period for this manuscript open until December 1, mercury as a heavy metal is a life-threatening factor 2017 on GJESM website at the “Show Article”. to human health (Järup, 2003). Prokaryotes establish a M. Noroozi et al. large part of the atmosphere and are less considerable m above the sea level (Fig. 1). Gavkhuni wetland than other organisms (Ghiasian et al., 2017). is an international wetland, registered in Ramsar Moderately halophilic bacteria are in a diverse group Convention in 1975. It has maximum width of 50 km of microorganisms which are able to grow in light- and length of 25 km. This wetland has an area of about salt media and tolerate high salt concentrations. These 470 km2 depending on water entry to the wetland, as bacteria are beneficial for the bioremediation of toxic its space reduces in summer because of inflow and metals from contaminated milieu (Amoozegar et al., higher evaporation rate and expands considerably in 2008). The mer operon has two relevant genes: merR humid seasons (Gohari et al.,2013). and merA genes. The former regulates detoxification enzymes and the latter encodes mercury ion reductase, Isolation of microorganisms, culture media and both of which have associated roles with prokaryotic growth conditions physiology (Barkay and Irene, 2005). The mercuric After preparing serial dilutions, the samples were reductase (MR) enzyme modifies mercury toxicity cultured on nutrient agar medium (peptone 5g, yeast and mobility including inorganic Hg [Hg(II)] in extract 3g, agar 15g, dH2O 500 mL, and pH=7.5) nature. Bacteria and archaea have the merA gene to enriched by 5% (W/V) NaCl and 0.005 mM HgCl2 survive against high amount of mercury (Freedman et and finally incubated at 34 °C for 48h with intense al., 2012). Mercuric ion detoxification in bacteria is shaking. After the growth and appearance of colonies, catalyzed through mercuric reductase containing FAD single colonies were cultured to produce pure strains. and is dependent on NAD(P)H (EC 1.16.1.1) (Boden Purification of colonies was performed at least in and Murrell, 2011). Extreme environments made both 5 successive cultures of isolates on the nutrient naturally or by human activities, e.g. polluted sites agar medium enriched by 5% (W/V) NaCl. The and mines, are attracting commercial interests and are strains were tested for morphological, physiological suitable bacterial studies on adaptation and evolution. and biochemical characteristics using standard (Héry et al., 2005). This study has been carried out in microbiological criteria, such as pigmentation, Gavkhuni wetland in Iran in 2014 in order to isolate form, colonial elevation and opacity under light and identify the moderately halophilic bacteria which microscopy. Gram staining was done and the result produce mercuric reductase. was approved by KOH test. The wet-mount method was used to examine the motility of the bacteria MATERIALS AND METHODS (Smibert and Krieg, 1994). The tolerance to a range Study area and sample collection of mercury ions was determined for the bacteria Initially soil samples were taken from Gavkhuni which had been isolated by agar dilution method. wetland. Gavkhuni wetland with geographical First, various concentrations of mercury (0.005 up to coordinates of 15’ 32o - 22’ 32o N and 45’ 52o - 59’ 5 mM) were added to flasks containing the required 52oE is located at the southeast of Isfahan, Iran, 30 amount of nutrient agar medium and 5% (W/V) NaCl. km away from the nearest town (Varzaneh) and 1470 Then, contents of the flasks were poured into eight- Fig.Fig. 1: 1: The The study study area 428 Global J. Environ. Sci. Manage., 3(4): 427-436, Autumn 2017 centimeter plates. Subsequently, 10 μl of the bacteria last, the constructed tree topology was drawn through suspension (1.5×108 cfu /ml) was poured on agar bootstrap assay of neighbor-joining data set according medium using a sampler. The plates were examined to 100 resampling attempts (Felsenstein, 1985). after incubation at 34 °C for 72 hours. Based on previous studies on heavy metals-resistant halophilic Mercuric reductase assay bacteria, the isolates which can grow in a medium Luria-Bertani medium with 10 μM HgCl2 was containing 0.1 mM Hg are considered resistant. used to dilute (1:20) overnight cultures incubated in The lowest concentration of metal compounds that shaking incubator at optimal temperatures with optical completely inhibited the bacterial growth was named density at 660 nm of 0.4. Incubation continued for 10 MIC (Minimum Inhibitory Concentration). All m after adding final concentration of 10 μM HgCl2 to experiments were performed twice (Abou-Shanab et induce bacterial cells. After this period, the obtained al., 2007). suspensions were chilled on ice and then centrifuged in pre-weighed tubes. Supernatants were discarded DNA extraction and amplification of 16S rRNA gene and the precipitated bacterial cells were re-suspended The identification process for mercury- in PBS, and after weighing were reserved at −20 °C up resistant soil bacteria was carried out by 16S to testing. Resultant precipitated bacterial cells were rRNA gene sequencing after extracting bacterial poured into a buffer (sodium phosphate 20mM (pH genomic DNA from the cultures grown in Luria- 7.5), 0.5 mM EDTA, and 1 mM β-mercaptoethanol) Bertani medium using Kit (Sigma). The DNA to prepare suspensions with about 200 mg/ml was amplified by universal 16SrRNA primers of concentration (fresh weight), and intermittent 27F(5’-AGAGTTTGATCMTGGCTCAG-3’) and sonication was utilized to cleave the bacterial cells on 1492R(5’-ACGGCTACCTTGTTACGA-3’) via PCR ice for 10 m. Broken cells were centrifuged at 14,000 method. The program of PCR amplification for this rpm and 4 °C for 30 m. Pellets were delivered on ice gene consisted of 96 °C (5 min) that was followed after removing the supernatant (Vetriani et al., 2005). through 30 cycles of 95°C (15 s), 49 °C (30 s) and 72 The subsequent assessments were conducted in 80 °C (1 min) and terminal expanse of 72°C (10 min). mM of sodium phosphate solution (pH 7.4), 1 mM Macrogen Co. (South Korea) determined 16S rRNA β-mercaptoethanol, 200 μM NADPH, and 100 μM gene sequencing, and the obtained data were edited HgCl2 (Fox and Walsh, 1982). Decreasing trend of on Chromas Pro bioinformatic software (Giri et al, A340 showed HgCl2-dependent oxidation of NADPH 2014). via spectrophotometer. One unit of activity means one μmol of NADPH oxidized per minute. Bradford merA phylogeny and bioinformatic analyses assay was used to calculate protein concentrations The merA gene of selected strain was amplified by in crude cell extractions.