جداسازی و تعیین توالی ژن مقاومت به جیوه در باکتری رائولتلا پلانتی کولا
محورهای موضوعی : میکروب شناسی محیطی
اشرف رضایی
1
(کارشناس ارشد، گروه میکروب شناسی، دانشکده علوم، واحد دامغان، دانشگاه آزاد اسلامی، دامغان)
مجید مقبلی
2
(استادیار، گروه میکروب شناسی، دانشکده علوم، واحد دامغان، دانشگاه آزاد اسلامی، دامغان)
کلید واژه: جیوه, واکنش زنجیره ای پلی مراز, ژن merA, رائولتلا پلانتی کولا,
چکیده مقاله :
سابقه و هدف: جیوه یکی از منابع آلوده کننده محیط زیست، گیاهان و انسان می باشد. میکروارگانیسم ها با مکانیسم های مختلف نسبت به جیوه مقاومند که اصلی ترین این مکانیسم ها احیای جیوه سمی به جیوه عنصری می باشد. این توانمندی با واسطه ژن های مختلف در باکتری ها می باشد. هدف از این تحقیق بررسی وجود ژن مقاومت به جیوه در باکتری رائولتلا پلانتی کولا از طریق واکنش زنجیره ای پلی مراز و تعیین توالی می باشد. مواد و روش ها: از باکتری رائولتلا پلانتی کولا که قبلا از پساب جدا شده بود، DNA ژنومی و پلاسمید جدا شد. سپس با استفاده از پرایمرهایی که بر اساس وجود ژن merA در باکتری کلبسیلا اکسی توکا طراحی شده بود واکنش زنجیره پلی مراز انجام پذیرفت. محصول به دست آمده تعیین توالی گردید و با استفاده از سایت NCBI، توالی مورد تجزیه و تحلیل قرار گرفت. یافته ها: با استفاده از واکنش زنجیره ای پلی مراز بر روی پلاسمید و DNA ژنومی جدا شده از باکتری، باند 1700 جفت بازی به دست آمد. از Blast توالی به دست آمده مشخص گردید که ژن تکثیر شده 99 درصد مشابه ژنmerA در باکتری های انتروباکتر کلوآکه، اشریشیا کلی، سراشیا مارسسنس، کلبسیلا اکسی توکا و آسینتوباکتر است. نتیجه گیری: برای اولین بار در این تحقیق مشخص گردید که رائولتلا پلانتی کولا جدا شده از منابع طبیعی ایران با داشتن ژن merA نسبت به جیوه مقاوم است. به همین دلیل این باکتری می تواند کاندید مناسبی به منظور استفاده در کاهش یا حذف جیوه در پساب های صنعتی باشد.
Background & Objectives: Mercury is one of the polluting sources of the environment, plants as well as human. Microorganisms are resistant to mercury through different mechanisms. The main mechanism involves reduction of the toxic mercury to the elemental form which is mediated through a variety of bacterial genes. The aim of this study is to investigate the presence of mercury resistance gene in Raoultella planticula bacteria using polymerase chain reaction (PCR) and sequencing methods. Materials & Methods: Genomic and plasmid DNA was extracted from wastewater R. planticula isolates. PCR was set up with primers designed based on the presence of mer A gene in Klebsiella oxytoca. The PCR products were sequenced and analyzed using NCBI database. Results: Using PCR on plasmid and genomic bacterial DNA, a 1700 bps bond was obtained. Blasting the sequence, it was found that the amplified gene has 99% sequence homology to merA gene in Enterobacter cloacae, E. coli, Serratia marcescens, Klebsiella oxytoca and Acinetobacter. Conclusion: For the first time in this study, we found that Raoultella planticula isolated from Iran natural sources are resistant to mercury due to the presence of merA gene. Therefore, R. planticula can be considered an appropriate candidate to reduce or remove mercury from industrial wastewaters.
1. Wali F, Kepel B, Yusuf I, Badaruddin F, Natsir R, Retnoningrum D. Isolation and characterization of partial sequence of merA gene from mercury resistant bacterium Klebsiella pneumoniae isolated from Sario River Estuary Manado. Res J Environ Earth Sci. 2014; 6(3): 156-160.
2. Jan AT, Murtaza I, Ali A, Rizwanul Haq QM. Mercury pollution: An emerging problem and potential bacterial remediation strategies. J Microbiol Biotechnol. 2009; 25: 1529-1537.
3. Summers AO, Silver S. Mercury resistance in a plasmid-bearing strain of Escherichia coli. J Bacteriol. 1972 ; 112: 1228-1236.
4. Poulain AJ, Ni Chadhain SM, Ariya PA. Potential for mercury reduction by microbes in the high Arctic. Appl Environ Microbiol. 2007; 73(11): 3769.
5. Griffin HG, Foster TJ, Silver S, Misra TK. Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358. Proc Natl Acad Sci USA. 1987; 84: 3112-3116.
6. Bhriain NN. Foster TJ. Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100. Gene. 1986; 42(3): 323-330.
7. Misra TK, Brown NL, Fritzinger DC, Pridmore RD, Barnes WM, Haberstroh L, Silver S. The mercuric-ion resistance operons of plasmid R100 and transposon Tn501: The beginning of the operon including the regulatory region and the first two structural genes. Proc Natl Acad Sci USA. 1984; 81: 5975-5979.
8. Liebert CA, Hall RM, Summers AO. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol. 1999; 63: 507-522.
9. Lu Y, Wilkins E. Heavy metal removal by caustic treated yeast immobilized in alginat. J Haz Mat. 1996; 49(2-3): 165-179.
10. Schelert J, Divixt V, Hoang V, Simbahan J, Drozda M, Blum P. Occurrence and characterization of mercury resistance in the hyper thermophilic archaeon sulfolobus solfataricus by use gene disruption. J Bacteriol. 2004; 186(2): 427-437.
11. Adeniji A. Bioremediation of arsenic, chromium, lead and mercury. NSCEP. Available at: http://nepis.epa.gov. (2004).
12. Moghbeli M, Shakeri F, Hashemi-Moghaddam H. Separation of mercury resistant bacteria from wastewater of milk, detergent and ceramic industry. J Chemi Health Risks. 2011; 1(1): 19-22.
13. Wei G, Fan L, Zhu W, Fu Y, Yu J, Tang M. Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailing in China. J Hazard Mat. 2009; 162(1): 50-56.
14. Maiti A, Bhattacharyya S. Isolation and characterization of mercury resistant bacteria from Haldia river sediments. IOSR J Environ Sci Toxic Food Tech. 2013; 5(3): 23-28.
15. Akhavan Sepahy A, Sharifian S, Zolfaghari A, Khalili Dormani M. Evaluation of resistance in intestinal coli forms isolated from industrial wastewater, domestic wastewater and various parts of the wastewater treatment system of Arak city. Cell Mole Res J. 2014; 27(2): 167-178. [In Persian]
16. Kafilzadeh F, Aram M, Sharifi A, Naghmachi M. Isolation and survey growth kinetics of mercury resistant bacteria in Lake Maharloo. Iran J Med Microbiol. 2012; 6(1&2): 28-38. [In Persian]
17. Lipthay JRd, Rasmussen LD, Oregaard G, Simonsen K, Bahl MI, Kroer N. The adaptive potential of subsurface microbial communities to mercury. FEMS Microbiol Ecol. 2007; 3(2): 1766-1770.
18. Oregaard G, Sørensen SJ. High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA). Int Soc Microbiol. 2007; 1: 453-467.
19. Adriana SM, Michele SDJ, Michele L, Josino CM, Ana Luzia LF, Paulo Rubens GB. A conservative region of the mercuric reeducates gene (merA) as a molecular marker of bacterial mercury resistance. Braz J Microbiol. 2008; 39(2): 173-179.
20. Kargar M, Jahromi MZ, Najafian M, Khajeaian P, Nahavandi R, Jahromi SR, Firoozinia M. Identification and molecular analysis of mercury resistant bacteria in Kor River, Iran. Afr J Biotechnol. 2012; 11(25): 6710-6717.
21. Zeyaullah Md, Islam B, Ali A. Isolation, identification and PCR amplification of merA gene from highly mercury polluted Yamuna river. Afric J Biotech. 2010; 9(24): 3510-3514.
22. Moller AK, Søborg DA, Abu Al-Soud W, Sørensen SJ, Kroer N. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyro sequencing of 16S rRNA genes and cultivation. Polar Res. 2013; 32: 17390.
23. Moller Mk, Barkay T, Hansen MA, Norman A, Hansen LH. Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine. FEMS Microbiol Ecol. 2013; 87: 52-63.
24. Tett A, Spiers AJ, Crossman LC. Sequence-based analysis of pQBR103; a representative of a unique, transfer proficient mega plasmid resident in the microbial community of sugar beet. ISME J. 2007; 1: 331-340.
25. Musovic S, Oregaard G, Kroer N, Sørensen SJ. Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among Gram-positive and Gram-negative bacteria indigenous to the barley rhizosphere. Appl Environ Microbiol 2006; 72: 6687-6692.
26. Miteva V, Lantz S, Brenchley J. Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients. Extremophiles. 2008; 12: 441-449.
27. Chien MF, Lin KH, Chang JE, Huang CC, Endo G, Suzuki S. Interdisciplinary studies on environmental chemistry biological responses to contaminants, from molecular to community level, interdisciplinary studies on environmental chemistry. 2003; 3: 31-36.
28. Narita M, Chiba K, Nishizawa H, Ishii H, Huang CC, Kawabata Z, Silver S, Endo G. Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay. FEMS Microbiol. Letters. 2003; 226(2): 415.
_||_1. Wali F, Kepel B, Yusuf I, Badaruddin F, Natsir R, Retnoningrum D. Isolation and characterization of partial sequence of merA gene from mercury resistant bacterium Klebsiella pneumoniae isolated from Sario River Estuary Manado. Res J Environ Earth Sci. 2014; 6(3): 156-160.
2. Jan AT, Murtaza I, Ali A, Rizwanul Haq QM. Mercury pollution: An emerging problem and potential bacterial remediation strategies. J Microbiol Biotechnol. 2009; 25: 1529-1537.
3. Summers AO, Silver S. Mercury resistance in a plasmid-bearing strain of Escherichia coli. J Bacteriol. 1972 ; 112: 1228-1236.
4. Poulain AJ, Ni Chadhain SM, Ariya PA. Potential for mercury reduction by microbes in the high Arctic. Appl Environ Microbiol. 2007; 73(11): 3769.
5. Griffin HG, Foster TJ, Silver S, Misra TK. Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358. Proc Natl Acad Sci USA. 1987; 84: 3112-3116.
6. Bhriain NN. Foster TJ. Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100. Gene. 1986; 42(3): 323-330.
7. Misra TK, Brown NL, Fritzinger DC, Pridmore RD, Barnes WM, Haberstroh L, Silver S. The mercuric-ion resistance operons of plasmid R100 and transposon Tn501: The beginning of the operon including the regulatory region and the first two structural genes. Proc Natl Acad Sci USA. 1984; 81: 5975-5979.
8. Liebert CA, Hall RM, Summers AO. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol. 1999; 63: 507-522.
9. Lu Y, Wilkins E. Heavy metal removal by caustic treated yeast immobilized in alginat. J Haz Mat. 1996; 49(2-3): 165-179.
10. Schelert J, Divixt V, Hoang V, Simbahan J, Drozda M, Blum P. Occurrence and characterization of mercury resistance in the hyper thermophilic archaeon sulfolobus solfataricus by use gene disruption. J Bacteriol. 2004; 186(2): 427-437.
11. Adeniji A. Bioremediation of arsenic, chromium, lead and mercury. NSCEP. Available at: http://nepis.epa.gov. (2004).
12. Moghbeli M, Shakeri F, Hashemi-Moghaddam H. Separation of mercury resistant bacteria from wastewater of milk, detergent and ceramic industry. J Chemi Health Risks. 2011; 1(1): 19-22.
13. Wei G, Fan L, Zhu W, Fu Y, Yu J, Tang M. Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailing in China. J Hazard Mat. 2009; 162(1): 50-56.
14. Maiti A, Bhattacharyya S. Isolation and characterization of mercury resistant bacteria from Haldia river sediments. IOSR J Environ Sci Toxic Food Tech. 2013; 5(3): 23-28.
15. Akhavan Sepahy A, Sharifian S, Zolfaghari A, Khalili Dormani M. Evaluation of resistance in intestinal coli forms isolated from industrial wastewater, domestic wastewater and various parts of the wastewater treatment system of Arak city. Cell Mole Res J. 2014; 27(2): 167-178. [In Persian]
16. Kafilzadeh F, Aram M, Sharifi A, Naghmachi M. Isolation and survey growth kinetics of mercury resistant bacteria in Lake Maharloo. Iran J Med Microbiol. 2012; 6(1&2): 28-38. [In Persian]
17. Lipthay JRd, Rasmussen LD, Oregaard G, Simonsen K, Bahl MI, Kroer N. The adaptive potential of subsurface microbial communities to mercury. FEMS Microbiol Ecol. 2007; 3(2): 1766-1770.
18. Oregaard G, Sørensen SJ. High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA). Int Soc Microbiol. 2007; 1: 453-467.
19. Adriana SM, Michele SDJ, Michele L, Josino CM, Ana Luzia LF, Paulo Rubens GB. A conservative region of the mercuric reeducates gene (merA) as a molecular marker of bacterial mercury resistance. Braz J Microbiol. 2008; 39(2): 173-179.
20. Kargar M, Jahromi MZ, Najafian M, Khajeaian P, Nahavandi R, Jahromi SR, Firoozinia M. Identification and molecular analysis of mercury resistant bacteria in Kor River, Iran. Afr J Biotechnol. 2012; 11(25): 6710-6717.
21. Zeyaullah Md, Islam B, Ali A. Isolation, identification and PCR amplification of merA gene from highly mercury polluted Yamuna river. Afric J Biotech. 2010; 9(24): 3510-3514.
22. Moller AK, Søborg DA, Abu Al-Soud W, Sørensen SJ, Kroer N. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyro sequencing of 16S rRNA genes and cultivation. Polar Res. 2013; 32: 17390.
23. Moller Mk, Barkay T, Hansen MA, Norman A, Hansen LH. Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine. FEMS Microbiol Ecol. 2013; 87: 52-63.
24. Tett A, Spiers AJ, Crossman LC. Sequence-based analysis of pQBR103; a representative of a unique, transfer proficient mega plasmid resident in the microbial community of sugar beet. ISME J. 2007; 1: 331-340.
25. Musovic S, Oregaard G, Kroer N, Sørensen SJ. Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among Gram-positive and Gram-negative bacteria indigenous to the barley rhizosphere. Appl Environ Microbiol 2006; 72: 6687-6692.
26. Miteva V, Lantz S, Brenchley J. Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients. Extremophiles. 2008; 12: 441-449.
27. Chien MF, Lin KH, Chang JE, Huang CC, Endo G, Suzuki S. Interdisciplinary studies on environmental chemistry biological responses to contaminants, from molecular to community level, interdisciplinary studies on environmental chemistry. 2003; 3: 31-36.
28. Narita M, Chiba K, Nishizawa H, Ishii H, Huang CC, Kawabata Z, Silver S, Endo G. Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay. FEMS Microbiol. Letters. 2003; 226(2): 415.