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Applied and Environmental Microbiology, July 2003, p . 4332-4336, Vol . 69, No . 7

Molecular Phylogenetic Exploration of Bacterial Diversity in a Bakreshwar (India) Hot Spring and Culture of Shewanella-Related Thermophiles

Dhritiman Ghosh,1 Bijay Bal,2 V . K . Kashyap,3 and Subrata Pal1*

Department of Life Science and Biotechnology, Jadavpur University, Calcutta 700 032,1 Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Calcutta 700 064,2 Central Forensic Science Laboratory, Calcutta 700 014, India3

Received 14 August 2002/ Accepted 2 April 2003


   ABSTRACT

 
The bacterial diversity of a hot spring in Bakreshwar, India, was investigated by a culture-independent approach . 16S ribosomal DNA clones derived from the sediment samples were found to be associated with gamma-Proteobacteria, cyanobacteria, and green nonsulfur and low-GC gram-positive bacteria . The first of the above phylotypes cobranches with Shewanella, a well-known iron reducer . This phylogenetic correlation has been exploited to develop culture conditions for thermophilic iron-reducing microorganisms .


   INTRODUCTION

 
Microbial metal reduction has become a subject of intensive investigation as a result of its overwhelming environmental significance (4) . This has led to the isolation and identification of a number of metal-reducing bacteria . Bacterial species identified as dissimilatory metal reducers include facultative anaerobes such as Shewanella oneidensis and Shewanella alga as well as strict anaerobes like Geobacter metallireducens and Desulfovibrio sp . (18) . Shewanella, a gram-negative bacterium, can use a wide range of electron acceptors, including fumarate, trimethylamine N-oxide, dimethyl sulfoxide, nitrate, nitrite, thiosulfate, and sulfite, as well as insoluble acceptors, such as metal oxides or oxyhydroxides (14, 16) . It is widely distributed in freshwater and marine environments (18, 19, 21, 23) . A psychrophilic and moderately barophilic strain of Shewanella violacea has been discovered in deep-sea sediments (20) . However, to date no thermophilic strain related to Shewanella has been reported .

A number of thermophiles and hyperthermophiles have been isolated from samples of hot sediments, mud, rocks, soils, and waters . Hot environments have been searched also for metal reducers . Certain hyperthermophiles such as Thermotoga maritima are known to grow as respiratory organisms when Fe(III) is provided as an electron acceptor (27) . Furthermore, Pyrobaculum islandicum has been shown to reduce U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV) at 100°C (10) .

Several hot springs in different regions of the Indian subcontinent have been known to geologists for many years (6, 9, 12) . However, their microbial diversity has not been explored by molecular phylogenetic approaches . In the present study, we have applied the 16S rRNA methodology (2) to determine the bacterial community structure of Agnikunda, a hot spring in Bakreshwar, situated in the state of West Bengal in India . The investigation has revealed the presence, among a few other bacteria, of novel nonmarine, thermophilic relatives of Shewanella (formerly Alteromonas), a well-known iron reducer . Subsequently, iron-reducing enrichment culture to cultivate such organisms has been developed .


   Hot spring sediment DNA.

 
Samples were collected from the hot spring Agnikunda at Bakreshwar, in the Birbhum district of West Bengal in India . The surface temperature of the sediment varied between 66 and 69°C . The pH of the water was measured as 9.1 to 9.3 . The sediment contained 1.1 to 1.5% organic carbon and, interestingly, 280 to 422 µmol of reducible Fe(III) and 280 to 600 µmol of reduced iron per g of wet sediment . Initially, DNA extracted from the hot spring sediments by a direct lysis procedure (3, 29) could not be amplified in a PCR with Taq DNA polymerase, possibly due to the presence of high humic acid contamination in the samples (30) . Successive washes with buffers differing in EDTA concentration prior to lysis (28) enabled the purification of DNA having A260/A280 between 1.67 and 1.79 . Such DNA could be amplified successfully . The EDTA wash procedure, however, yielded only 1 to 3 µg of DNA from 4 g of wet sediment .


   Analysis of 16S rRNA genes.

 
In order to analyze the bacterial diversity in Agnikunda sediments, 16S ribosomal DNA (rDNA) libraries were constructed . Fifty nanograms of the total community DNA was amplified with bacterium-specific forward primer 5'-AGA GTT TGA ACA TGG CTG-3' (S-D-Bact-0027-a-S-18) and reverse primer 5'-CTA GCG ATT CCG ACT TCA-3' (S-D-Bact-1327-a-A-18) (1) . The numbers refer to the positions in the Escherichia coli 16S rRNA (5) . Reaction mixtures were incubated in a thermal cycler (GeneAmp 2400 PCR system; PE Applied Biosystems, Norwalk, Conn.) for an initial denaturation at 94°C for 2 min followed by 40 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min . Amplified DNAs were purified by the spin column method (Wizard PCR Prep DNA purification system; Promega Corp., Madison, Wis.) . The purified DNAs were cloned directly by the TA cloning method (13) with a pGEM-T Easy Vector System II kit (Promega) . Twenty-five positive clones were sequenced using either vector-specific primers or the same PCR primers . Homology search with the BLAST system showed that the hot spring sediment clones could be classified into four major phylotypes . Ten of the clones corresponded to the gamma subdivision of the Proteobacteria, eight were affiliated with cyanobacteria, three belonged to the green nonsulfur group, and four were low-GC gram-positive bacteria (Table 1) . All the sequences were checked for the presence of chimeric sequences by using the CHECK_CHIMERA program (available at http://rdp.cme.msu.edu) . No such chimeras were, however, detected .


TABLE 1 . Summary of the bacterial 16S rDNA clone sequences identified in Agnikunda (AKB) sediment

 

   Phylogenetic analysis.

 
The gamma-proteobacterial sequences retrieved from Agnikunda sediment were aligned with sequences in the small-subunit rRNA database of the RDP server by using the Clustal W 1.6 program (26) . Phylogenetic analysis was restricted to nucleotide positions that could be unambiguously aligned in all the sequences . A phylogenetic tree was constructed using a maximum likelihood algorithm (7) with 100-bootstrap resampling (8) (Fig . 1) . Among the 10 proteobacterial clones there were eight unique sequences . Three of the 16S rRNA sequences are represented by AKB03 . In the tree, one clone (AKB13) clusters within the Shewanella genus . This clone shows 95% nucleotide identity to S . alga (U91544) (Table 1) . The result is consistent with the phylogenetic position of this sequence assigned by the RDP at http://rdp.cme.msu.edu/cgis/hierarchy_preview.cgi . The seven other clones (AKB03, AKB06, AKB07, AKB08, AKB09, AKB10, and AKB11) group separately and form a cobranch with species in the Shewanella cluster originating from a common node with a bootstrap value of 50 (Fig . 1) . The sequence identity of these clones with different Shewanella species varied between 86 and 91% (Table 1) . It may be noted here that the RDP has placed at least AKB11 in the same family as Shewanella, Alteromona-daceae (http://rdp.cme.msu.edu/cgis/hierarchy_preview.cgi).The results obtained with the maximum likelihood algorithm were also verified by using the neighbor joining DNA distance (22) and maximum parsimony (25) treeing methods (data not shown) .


FIG . 1 . Phylogenetic tree deduced from the gamma-proteobacterial 16S rDNA of hot spring sediment clones by maximum likelihood algorithm . Reference sequences were chosen to represent the broadest diversity of Bacteria . Aquifex pyrophilus and Chloroflexus aggregans were used as outgroups for the analysis . Division- and subdivision-level groupings are either bracketed or marked with a horizontal line at the right of the figure . Branch points supported (bootstrap values) are indicated at the branch points of the nodes of the tree . A scale is shown below the tree to indicate the branch length.

 

   Enrichment culture and Fe(III) reduction.

 
A number of Shewanella strains are known to use for anaerobic respiration a wide variety of electron acceptors including metal oxides and hydroxides (16, 17) . In an attempt to cultivate the Shewanella-like iron-reducing microorganisms present in the hot spring sediments, enrichment medium containing (per liter of deionized water) 0.33 g of KH2PO4, 0.33 g of NaCl, 0.33 g of KCl, 0.6 g of NaH2PO4, and 2.5 g of Na2CO3 was prepared . Yeast extract (0.01%, wt/vol) was added as a vitamin supplement, and 10 mM pyruvate was added as an electron donor . The pH was adjusted to 7.0 at 25°C with 10% (wt/vol) NaOH . Amorphous Fe(III) oxyhydroxide, used as the electron acceptor at ca . 90 mmol of Fe(III) per liter, was synthesized by titrating a solution of FeCl3 with 10% (wt/vol) NaOH to pH 9.0 (24) . Cultures were grown in 10 ml of medium in Hungate tubes under an atmosphere of CO2 (100%) (24) at 66°C . Such conditions favored growth as well as Fe(III) reduction with time (Fig . 2) . The growth continued until the 14th day . There was a concomitant increase in the concentration of reduced iron in the medium . The maximum Fe(III) reduction was found to be 96% in 19-day-old cultures . No growth could be observed when either Fe(III) or pyruvate was omitted (Fig . 2) or pyruvate was replaced with citrate or acetate (data not shown) .


FIG . 2 . Growth of Shewanella-like consortium at 66°C and Fe(III) reduction in a medium with pyruvate as the electron donor and poorly crystalline Fe(III) oxide as the electron acceptor . The medium also contained 0.01% yeast extract . The results are the means of triplicate cultures . OD540, optical density at 540 nm.

 
Twelve 16S rDNA clones were retrieved from the enrichment culture by using the same set of PCR primers as above (27F and 1327R) . From these, six unique sequences were revealed . These sequences were aligned with those retrieved directly from the hot spring sediment . Two, four, two, and one enrichment clone completely matched with four proteobacterial clones, AKB03, AKB06, AKB07, and AKB13, respectively . In the cases of the other two sequences (representing three clones) there were some deviations which could be due to statistical error or bias caused by PCR . Our results thus validate the earlier suggestions made by Lovley and his coworkers (27) that isolation of as-yet-uncultured thermophiles or hyperthermophiles with medium containing Fe(III) as the electron acceptor could be a productive strategy for culturing these organisms . The relative abundance of gamma-Proteobacteria in both sediment and enrichment culture samples was determined by hybridization to a fluorescently labeled oligonucleotide probe specific for 23S rRNA of the subdivision . Approximately 30% of the microorganisms in the sediment were gamma-Proteobacteria, whereas in the enrichment culture the proportion was nearly 90% (data not shown) .


   Concluding remarks.

 
The discovery of Shewanella-like thermophilic bacteria is interesting from the standpoints of both the understanding of molecular genetics of metal reduction at higher temperatures and its biotechnological applications (11) . Although a number of dissimilatory metal reducers, mesophilic and thermophilic, are already known, the molecular genetics of metal reduction by different Shewanella strains has been extensively studied . Cell fractionation studies of S . oneidensis MR-1 demonstrated the presence of ferric reductase activity in the outer membrane as well as the inner membrane of anaerobically grown cells (15) . mtrB, a gene that encodes an outer membrane involved in Fe(III) and Mn(IV) reduction, has been isolated and sequenced (4) . Myers and Myers (17) have further discovered in S . oneidensis MR-1 outer membrane cytochrome genes omcA and omcB mutations which decrease the cells' ability to reduce Mn(IV) and not Fe(III) . Identification and a comparative analysis of similar genes in the thermophilic counterpart may provide a molecular insight into the mechanism of metal reduction at high temperatures . Work is in progress to isolate and characterize pure cultures of such microorganisms .

 


   ACKNOWLEDGMENTS

 
We express our deep gratitude to Binayak Dutta-Roy, who has been the main inspiration behind this work . We also thank Diti Chatterjee and Brajadulal Chattopadhyay of Jadavpur University and Ranjan Datta of the Central Forensic Science Laboratory for their technical help and cooperation .

This work was supported by a grant from the BRNS, Department of Atomic Energy, Government of India (sanction no . 97/37/31-BRNS/996), and a grant from the Department of Science and Technology, Government of India (SR/FIST/LS II-093/2000) .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Life Science and Biotechnology, Jadavpur University, Calcutta 700 032, India . Phone: 91-33-2414-6710 . Fax: 91-33-2414-6584 . E-mail: subrata_p{at}hotmail.com


   REFERENCES

 

  1. Alm, E . W., D . B . Oerther, N . Larsen, D . A . Stahl, and L . Raskin. 1996 . The oligonucleotide probe database . Appl . Environ . Microbiol . 62:3557-3559.
  2. Amann, R . I., W . Ludwig, and K.-H . Schleifer. 1995 . Phylogenetic identification and in situ detection of individual microbial cells without cultivation . Microbiol . Rev . 59:143-169.
  3. Barns, S . M., R . E . Fundyga, M . W . Jeffries, and N . R . Pace. 1994 . Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment . Proc . Natl . Acad . Sci . USA 91:1609-1613.
  4. Beliaev, A . S., and D . A . Saffarini. 1998 . Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction . J . Bacteriol . 180:6292-6297.
  5. Brosius, J., J . L . Palmer, J . P . Kennedy, and H . F . Noller. 1978 . Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli . Proc . Natl . Acad . Sci . USA 75:4801-4805.
  6. Chatterjee, G . C., and S . K . Guha. 1968 . The problems of origin of high temperature springs of India, p . 141-149 . In Proceedings of the 23rd International Geological Congress, vol . 17.
  7. Felsenstein, J. 1981 . Evolutionary trees from DNA sequences: a maximum likelihood approach . J . Mol . Evol . 17:368-376.
  8. Felsenstein, J. 1985 . Confidence limits on phylogenies: an approach using the bootstrap . Evolution 39:783-791.
  9. Gupta, M . L., H . Narain, and V . K . Saxena. 1975 . Geochemistry of thermal waters from various geothermal provinces of India, p . 47-58 . In Proceedings of the Grenoble Symposium of the International Association of Hydrological Sciences . IAHS Press, Institute of Hydrology, Wallingford, Oxfordshire, United Kingdom.
  10. Kashefi, K., and D . R . Lovley. 2000 . Reduction of Fe(III), Mn(IV), and toxic metals at 100°C by Pyrobaculum islandicum . Appl . Environ . Microbiol . 66:1050-1056.
  11. Lovley, D . R., and J . D . Coates. 2000 . Novel forms of anaerobic respiration of environmental relevance . Curr . Opin . Microbiol . 3:252-256.
  12. Majumdar, R . K., N . Majumdar, and A . L . Mukherjee. 2000 . Geoelectric investigations in Bakreswar geothermal area, West Bengal, India . J . Appl . Geophys . 45:187-202.
  13. Marchuk, D., M . Drumm, A . Saulino, and F . C . Collins. 1991 . Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products . Nucleic Acids Res . 19:1154.
  14. Moser, D., and K . Nealson. 1996 . Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction . Appl . Environ . Microbiol . 62:2100-2105.
  15. Myers, C., and J . Myers. 1993 . Ferric reductase is associated with the membranes of anaerobically grown Shewanella putrefaciens MR-1 . FEMS Microbiol . Lett . 108:15-22.
  16. Myers, C., and K . Nealson. 1988 . Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor . Science 240:1319-1321.
  17. Myers, J . M., and C . R . Myers. 2001 . Role of outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in the reduction of manganese dioxide . Appl . Environ . Microbiol . 67:260-269.
  18. Nealson, K . H., and D . Saffarini. 1994 . Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation . Annu . Rev . Microbiol . 48:311-343.
  19. Nealson, K . H., C . R . Myers, and B . Wimpee. 1991 . Isolation and identification of manganese reducing bacteria, and estimates of microbial manganese reducing potential in the Black Sea . Deep Sea Res . 38:S907-S920.
  20. Nogi, Y., C . Kato, and K . Horikoshi. 1998 . Taxonomic studies of deep-sea barophilic Shewanella strains and description of Shewanella violacea sp . nov . Arch . Microbiol . 170:331-338.
  21. Obuekwe, C., D . Westlake, J . Plambeck, and F . Cook. 1981 . Corrosion of mild steel in cultures of ferric iron reducing bacteria isolated from crude oil . II . Mechanism of anodic depolarization . Corrosion (Houston) 37:632-637.
  22. Saitou, N., and M . Nei. 1987 . The neighbor-joining method: a new method for reconstructing phylogenetic trees . Mol . Biol . Evol . 4:406-425.
  23. Semple, K., and D . Westlake. 1987 . Characterization of iron-reducing Alteromonas putrefaciens strains from oil field fluids . Can . J . Microbiol . 33:366-371.
  24. Slobodkin, A . I., T . P . Tourova, B . B . Kuznetsov, N . A . Kostrikina, N . A . Chernyh, and E . A . Bonch-Osmolovskaya. 1999 . Thermoanaerobacter siderophilus sp . nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium . Int . J . Syst . Bacteriol . 49:1471-1478.
  25. Swofford, D . L., G . J . Olsen, P . J . Waddell, and D . M . Hillis. 1996 . Phylogenetic inference, p . 407-514 . In D . M . Hillis, C . Moritz, and B . K . Mable (ed.), Molecular systematics . Sinauer Associates, Inc., Sunderland, Mass.
  26. Thompson, J . D., D . G . Higgins, and T . J . Gibson. 1994 . CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice . Nucleic Acids Res . 22:4673-4680.
  27. Vargas, M., K . Kashefi, E . L . Blunt-Harris, and D . R . Lovley. 1998 . Microbiological evidence for Fe(III) reduction on early Earth . Nature 395:65-67.
  28. Watson, R . J., and B . Blackwell. 2000 . Purification and characterization of a common soil component which inhibits the polymerase chain reaction . Can . J . Microbiol . 46:633-642.
  29. Yamamoto, H., A . Hiraishi, K . Kato, H . X . Chiura, Y . Maki, and A . Shimizu. 1998 . Phylogenetic evidence for the existence of novel thermophilic bacteria in hot spring sulfur-turf microbial mats in Japan . Appl . Environ . Microbiol . 64:1680-1687.
  30. Zhou, J., M . A . Bruns, and J . Tiedje. 1996 . DNA recovery from soils of diverse composition . Appl . Environ . Microbiol . 62:316-322.

 

 

 

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