تنوع مولکولی باکتری ها و آرکی های هتروتروف غار نمکدان قشم
محورهای موضوعی : میکروب شناسی محیطیمحبوبه دارابی 1 , محمد علی آموزگار 2 , ملیحه مهرشاد 3 , نینا زمانی 4 , سید ابوالحسن شاهزاده فاضلی 5 , محمود شوندی 6
1 - کارشناس ارشد، دانشگاه تهران، تهران
2 - استاد، دانشگاه تهران، تهران
3 - دکتری، دانشگاه تهران، تهران
4 - کارشناس ارشد، دانشگاه تهران، تهران
5 - دانشیار، مرکز ملی ذخایر ژنتیکی و زیستی ایران، تهران
6 - استادیار، گروه میکروب شناسی و بیوتکنولوژی، پژوهشگاه صنعت نفت، تهران
کلید واژه: تنوع زیستی, باکتری های نمک دوست, آرکی های نمک دوست, غار نمکدان قشم,
چکیده مقاله :
سابقه و هدف: با توجه به تنوع بالا، کاربردهای زیست فناوری و نقش موثر میکروارگانیسم ها در ایجاد و حفظ تعادل زیست بوم، کسب اطلاعات و توجه به تنوع زیستی میکروارگانیسم ها بسیار مورد نیاز است. در این میان، باکتری‌ها و آرکی های نمک‌دوست نیز به دلیل اهمیت اقتصادی و شرایط خاص اکولوژیکی زیست بوم شان مورد توجه می باشند. این مطالعه با هدف بررسی تنوع باکتری ها و آرکی های هتروتروف غار نمکدان قشم انجام شد.مواد و روش ها: این پژوهش به صورت مقطعی با نمونه برداری از غار نمکدان قشم در آبان ماه سال 1392 انجام شد. تنوع میکروارگانیسم های هوازی هتروتروف ساکن غار با استفاده از روش کشت مورد بررسی قرار گرفت. باکتری ها و آرکی های هالوفیل و هالوتولرانت در شرایط هوازی به ترتیب در دو محیط کشت Marine Agar و MGM جدا سازی شدند. جدایه ها براساس تفاوت های ریخت شناسی و ویژگی های بیوشیمیایی اولیه تفکیک شدند. در نهایت ژن rRNA 16S برای 32 سویه توالی یابی شد.یافته ها: بین 172 سویه خالص ژن rRNA 16S برای 27 سویه ترادف یابی شد که از نظر فیلوژنتیک آرکی ها در شاخه یوری آرکیوتا و درجنس های هالوباکتریوم، هالوآرکولا، هالوفراکس، هالوکوکوس و باکتری ها در شاخه های فرمی کیوتس و باکتریوئیدس و در جنس های آلیفودینی بیوس، باسیلوس، پارالیوباسیلوس،اکویی باسیلوس، پائنی باسیلوس قرار گرفتند. از بین این سویه ها 11 سویه شباهت کمتر از 98.7 درصد با نزدیک ترین سویه استاندارد داشتند که نقطه مرزی برای ارائه گونه جدید میکروبی محسوب می شود.نتیجه گیری: قرار گرفتن جدایه های شناسایی شده در شاخه ها و جنس های مختلف نشان دهنده تنوع بالای اکوسیستم غار نمکدان قشم از نظر میکروبی است. ارایه میکروارگانیسم های بومی در گونه ها و جنس های جدید و از اکوسیستم های منحصر به فرد با معرفی محتوای ژنتیکی جدید امکان دست یابی به فرایند ها و ژن های جدید بومی را فراهم می کند.
Background & Objectives: Given the high diversity, biotechnological applications and the effective role of bacteria in making and maintaining the ecosystem balance; biodiversity research are very important. Meanwhile, the halophilic bacteria and archaea have been considered because of their biotechnological importance and specific ecological condition. In this study, we investigated the diversity of heterotrophic bacteria and archaea of Namakdan cave in Qeshm Island. Materials & Methods: This cross-sectional study was carried out by sampling from Qeshm Namakdan cave in November 2013. The diversity of the cave heterotrophic aerobic bacteria was analyzed using the culture method. Halophilic and halotolerant bacteria and Archaea under aerobic conditions were isolated by MGM and Marine agar media, respectively. Isolates were separated according to morphological differences, and primary biochemical features. Finally, 16s rRNA sequencing was performed for 32 isolates.Results: Among 172 isolates 16S rRNA sequencing was carried out for 27 strains. Phylogenetic analysis placed archaea in the euryarchaeota division and Halococcus, Haloferax, Haloarcula, Halogeometricum genus branches and bacteria in Firmicutes and Bacteroides divisions and in Aliifodinibius, Paenibacillus, Aquibacillus, Paraliobacillus, and Bacillus genus branches. Among the sequenced isolates, 11 isolates showed less than 89.7% similarity to the standard species, which is considered as a borderline point to present new microbial species. Conclusion: Placing the identified isolates in different phylogenetic divisions and genus branches demonstrates the wide microbial diversity of Qeshm Namakdan cave ecosystem. Presenting native microorganisms in new species and genera from unique ecosystems by introducing new genetic content provides access to new native genes and pathways.
ahead. Mol Ecol. 2014; 14(2): 221-232.
2. Adams A, Tarmo A, Raadik, Christopher P, Burridge, Georges A. Global biodiversity
assessment and hypercriptic species complexes: more than one species of elephant in the
room?. Syst Biol. 2014; 63(4): 518-533.
3. Buttigieg PL, Alban Ramette A. Guide to statistical analysis in microbial ecology: a community
focused, living review of multyvariate data analyses. FEMS Microbial Ecol. 2014; 90(3):
543-550.
4. Stone M. Genetically enhanced Archaean challenges three-domain evolutionary tree. Bio Sci.
2015; 65(11): 1108.
5. Wolfe BE, Button JE, Santarelli M, Dutton RJ. Cheese rind communities provide tractable
systems for in situe and in vitro studies of microbial diversity. Cell. 2014; 158(2): 422-433.
6. Gillieson D. Caves: Processes. Development, Management. 2nd edition. Massachusetts, USA.
Blackwell; 2009.
7. Veni G, Hauwert N. Historical review and forward view of cave and karst research in Texas.
Geological Soc America. 2015; 516: 263-283.
8. Riquelme C, Marshall Hathaway JJ, Dapkevicius E, Miller AZ, Kooser A, Northup DE,
Jurado V, Fernandez O, Saiz-Jimenez C, Cheeptham N. Actinobacterial diversity in volcanic caves
and associated geomicrobiological interactions. Frontiers Microbiol. 2015; 1342(6):
467-483.
9. Romero A. Cave biology: life in darkness. First edition. Cambridge,UK. Cambridge
University Press. 2009.
10. Saiz-Jimene C. Microbiological and environmental issues in show caves. World J Microbiol
Biotechnol. 2012; 28(7): 2453-2464.
11. De Gruyter W. Microbial life of cave systems. First edition. Boston. Walter de Gruyter GmbH &
Co KG. 2015.
12. Desai MS, Assig K, Dattagupta S. Nitrogen fixation in distinct microbial niches within a
chemoautotrophy-driven cave ecosystem. ISME J. 2013; 7(12): 2411-2423.
13. Gray CJ, Engel AS. Microbial diversity and impact on carbonate geochemistry across a
changing geochemical gradient in a karst aquifer. ISME J. 2013; 7(2): 325-337.
14. Banerjee S, Joshi SR. Insights into cave architecture and the role of bacterial biofilm.
Proceedings of the National Academy of Sciences, India Section B: Biological Sci. 2013; 83(3):
277-290.
15. Ortiz M, Neilson JW, Nelson WM, Legatzki A, Byrne A, Yu Y, Wing RA, Soderlund CA, Pryor
BM, Pierson III LS. Profiling bacterial diversity and taxonomic composition on
speleothem surfaces in Kartchner Caverns, AZ. Microbiol Ecol. 2013; 65(2): 371-383.
16. Venarsky MP, Huntsman BM, Huryn AD, Benstead JP, Kuhajda BR. Quantitative food web
analysis supports the energy-limitation hypothesis in cave stream ecosystems. Oecologia. 2014;
176(3): 859-869.
17. Waltham T. Salt terrains of Iran. Geol Today. 2008; 24(5): 188-194.
18. Dyall-Smith M. The Halohandbook: Protocols for halobacterial genetics. Available at:
http://www.haloarchaea.com/resources/halohandbook. 2009.
19. Winn W, Allen S, Janda W, Konemen E, Procop G, Schreckenberger P, Woods G. Color
atlas and diagnostic microbiology. 6th edition. Baltimore. Lippincott Williams & Wilkins. 2006.
20. Rasooli M, Amoozegar MA, Akhavan Sepahy A, Babavalian H, Tebyanian H. Isolation,
identification and extracellular enzymatic activity of culturable extremely halophilic archaea and
bacteria of Inche Boroun wetland. Int Letter Natural Sci. 2016; 56: 40-51.
21. Murray R, Doetsch RN, Robinow C. Determinative and cytological light microscopy.
Method General Mol Bacteriol. 1994; 1: 22-41.
22. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. EzTaxon: a web-based tool for the
identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst
Evol Microbiol. 2007; 57(10): 2259-2261.
23. Ortiz M, Neilson JW, Nelson WM, Legatzki A, Byrne A, Yu Y, Pierson III LS. Profiling
bacterial diversity and taxonomic composition on speleothem surfaces in Kartchner Caverns, AZ.
Microbial Ecol. 2013; 65(2): 371-383.
24. Chang SJ, Blake RE, Stout LM, Kim SJ. Oxygen isotope, micro-textural and molecular
evidence for the role of microorganisms in formation of hydroxylapatite in limestone caves, South
Korea. Chem Geol. 2010; 276(3): 209-224.
25. Portillo MC, Saiz-Jimenez C, Gonzalez JM. Molecular characterization of total and
meta o ica acti e acteria communitie of “ ite co onization ” in t e A tamira a e ain.
Res Microbiol. 2009; 160(1): 41-47.
26. Macalady JL, Lyon EH, Koffman B, Albertson L.K, Meyer K, Galdenzi S, Mariani S.
Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system,
Italy. App Environ Microbiol. 2006; 72(8): 5596-5609.
27. Engel AS, Porter ML, Stern LA, Quinlan S, Bennett PC. Bacterial diversity and ecosystem
function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by
c emo it oautotro ic “ i on roteo acteria”. F M Micro io co . ; : -53.
28. Barton HA, Taylor MR, Pace NR. Molecular phylogenetic analysis of a bacterial community in an
oligotrophic cave environment. Geomicrobiol J. 2004; 21(1): 11-20.
29. Northup DE, Barns SM, Yu LE, Spilde MN, Schelble RT, Dano KE, Natvig DO. Diverse
microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves. Environ Microbiol. 2003; 5(11): 1071-1086.
30. Irannejad S, Akhavan-Sepahi A, Amoozegar MA, Tukmechi A, Motallabi Moghanjoghi AA.
Isolation and identification of halophilic bacteria from Urmia lake in Iran. Iran J Fisheries Sci.
2015; 14(1): 45-59.
31. Jookar Kashi F, Owlia P, Amoozegar MA, Yakhchali B, Kazemi B. Diversity of cultivable
microorganisms in the eastern part of Urmia salt lake, Iran. J Microbiol Biotechnol Food Sci. 2014;
4(1): 36-43.
32. Schabereiter-Gurtner C, Saiz-Jimenez C, Piñar G, Lubitz W, Rölleke S. Phylogenetic diversity of
bacteria associated with Paleolithic paintings and surrounding rock walls in two Spanish caves
(Llonin and La Garma). FEMS Microbiol Ecol. 2004; 47(2): 235-247.
33. Yildiz E, Ozcan B, Caliskan M. Isolation, characterization and phylogenetic analysis of
halophilic Archaea from a salt mine in central Anatolia (Turkey). Polish J Microbiol. 2012; 61:
111-117.
_||_
ahead. Mol Ecol. 2014; 14(2): 221-232.
2. Adams A, Tarmo A, Raadik, Christopher P, Burridge, Georges A. Global biodiversity
assessment and hypercriptic species complexes: more than one species of elephant in the
room?. Syst Biol. 2014; 63(4): 518-533.
3. Buttigieg PL, Alban Ramette A. Guide to statistical analysis in microbial ecology: a community
focused, living review of multyvariate data analyses. FEMS Microbial Ecol. 2014; 90(3):
543-550.
4. Stone M. Genetically enhanced Archaean challenges three-domain evolutionary tree. Bio Sci.
2015; 65(11): 1108.
5. Wolfe BE, Button JE, Santarelli M, Dutton RJ. Cheese rind communities provide tractable
systems for in situe and in vitro studies of microbial diversity. Cell. 2014; 158(2): 422-433.
6. Gillieson D. Caves: Processes. Development, Management. 2nd edition. Massachusetts, USA.
Blackwell; 2009.
7. Veni G, Hauwert N. Historical review and forward view of cave and karst research in Texas.
Geological Soc America. 2015; 516: 263-283.
8. Riquelme C, Marshall Hathaway JJ, Dapkevicius E, Miller AZ, Kooser A, Northup DE,
Jurado V, Fernandez O, Saiz-Jimenez C, Cheeptham N. Actinobacterial diversity in volcanic caves
and associated geomicrobiological interactions. Frontiers Microbiol. 2015; 1342(6):
467-483.
9. Romero A. Cave biology: life in darkness. First edition. Cambridge,UK. Cambridge
University Press. 2009.
10. Saiz-Jimene C. Microbiological and environmental issues in show caves. World J Microbiol
Biotechnol. 2012; 28(7): 2453-2464.
11. De Gruyter W. Microbial life of cave systems. First edition. Boston. Walter de Gruyter GmbH &
Co KG. 2015.
12. Desai MS, Assig K, Dattagupta S. Nitrogen fixation in distinct microbial niches within a
chemoautotrophy-driven cave ecosystem. ISME J. 2013; 7(12): 2411-2423.
13. Gray CJ, Engel AS. Microbial diversity and impact on carbonate geochemistry across a
changing geochemical gradient in a karst aquifer. ISME J. 2013; 7(2): 325-337.
14. Banerjee S, Joshi SR. Insights into cave architecture and the role of bacterial biofilm.
Proceedings of the National Academy of Sciences, India Section B: Biological Sci. 2013; 83(3):
277-290.
15. Ortiz M, Neilson JW, Nelson WM, Legatzki A, Byrne A, Yu Y, Wing RA, Soderlund CA, Pryor
BM, Pierson III LS. Profiling bacterial diversity and taxonomic composition on
speleothem surfaces in Kartchner Caverns, AZ. Microbiol Ecol. 2013; 65(2): 371-383.
16. Venarsky MP, Huntsman BM, Huryn AD, Benstead JP, Kuhajda BR. Quantitative food web
analysis supports the energy-limitation hypothesis in cave stream ecosystems. Oecologia. 2014;
176(3): 859-869.
17. Waltham T. Salt terrains of Iran. Geol Today. 2008; 24(5): 188-194.
18. Dyall-Smith M. The Halohandbook: Protocols for halobacterial genetics. Available at:
http://www.haloarchaea.com/resources/halohandbook. 2009.
19. Winn W, Allen S, Janda W, Konemen E, Procop G, Schreckenberger P, Woods G. Color
atlas and diagnostic microbiology. 6th edition. Baltimore. Lippincott Williams & Wilkins. 2006.
20. Rasooli M, Amoozegar MA, Akhavan Sepahy A, Babavalian H, Tebyanian H. Isolation,
identification and extracellular enzymatic activity of culturable extremely halophilic archaea and
bacteria of Inche Boroun wetland. Int Letter Natural Sci. 2016; 56: 40-51.
21. Murray R, Doetsch RN, Robinow C. Determinative and cytological light microscopy.
Method General Mol Bacteriol. 1994; 1: 22-41.
22. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. EzTaxon: a web-based tool for the
identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst
Evol Microbiol. 2007; 57(10): 2259-2261.
23. Ortiz M, Neilson JW, Nelson WM, Legatzki A, Byrne A, Yu Y, Pierson III LS. Profiling
bacterial diversity and taxonomic composition on speleothem surfaces in Kartchner Caverns, AZ.
Microbial Ecol. 2013; 65(2): 371-383.
24. Chang SJ, Blake RE, Stout LM, Kim SJ. Oxygen isotope, micro-textural and molecular
evidence for the role of microorganisms in formation of hydroxylapatite in limestone caves, South
Korea. Chem Geol. 2010; 276(3): 209-224.
25. Portillo MC, Saiz-Jimenez C, Gonzalez JM. Molecular characterization of total and
meta o ica acti e acteria communitie of “ ite co onization ” in t e A tamira a e ain.
Res Microbiol. 2009; 160(1): 41-47.
26. Macalady JL, Lyon EH, Koffman B, Albertson L.K, Meyer K, Galdenzi S, Mariani S.
Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system,
Italy. App Environ Microbiol. 2006; 72(8): 5596-5609.
27. Engel AS, Porter ML, Stern LA, Quinlan S, Bennett PC. Bacterial diversity and ecosystem
function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by
c emo it oautotro ic “ i on roteo acteria”. F M Micro io co . ; : -53.
28. Barton HA, Taylor MR, Pace NR. Molecular phylogenetic analysis of a bacterial community in an
oligotrophic cave environment. Geomicrobiol J. 2004; 21(1): 11-20.
29. Northup DE, Barns SM, Yu LE, Spilde MN, Schelble RT, Dano KE, Natvig DO. Diverse
microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves. Environ Microbiol. 2003; 5(11): 1071-1086.
30. Irannejad S, Akhavan-Sepahi A, Amoozegar MA, Tukmechi A, Motallabi Moghanjoghi AA.
Isolation and identification of halophilic bacteria from Urmia lake in Iran. Iran J Fisheries Sci.
2015; 14(1): 45-59.
31. Jookar Kashi F, Owlia P, Amoozegar MA, Yakhchali B, Kazemi B. Diversity of cultivable
microorganisms in the eastern part of Urmia salt lake, Iran. J Microbiol Biotechnol Food Sci. 2014;
4(1): 36-43.
32. Schabereiter-Gurtner C, Saiz-Jimenez C, Piñar G, Lubitz W, Rölleke S. Phylogenetic diversity of
bacteria associated with Paleolithic paintings and surrounding rock walls in two Spanish caves
(Llonin and La Garma). FEMS Microbiol Ecol. 2004; 47(2): 235-247.
33. Yildiz E, Ozcan B, Caliskan M. Isolation, characterization and phylogenetic analysis of
halophilic Archaea from a salt mine in central Anatolia (Turkey). Polish J Microbiol. 2012; 61:
111-117.