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Cold-Loving Bacteria from Antarctic and Arctic : Occurrence, Survival and Usefulness

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Cold-Loving Bacteria from Antarctic and Arctic : Occurrence, Survival and Usefulness COLD-LOVING BACTERIA FROM ANTARCTIC AND ARCTIC : OCCURRENCE, SURVIVAL AND USEFULNESS Dr S Shivaji CCMB, Hyderabad March, 2012 Extremophiles Deep sea vent Sea ice Thermophiles Cold loving microbes (psychrophiles) Soda lake Alkali-loving microbes (Alkaliphiles) Acid-loving microbes Natronobacterium (Acidophiles) gregoryi Haloferax volcanii Sulfolobus acidocaldarius Salt-loving microbes (Halophiles) Salt lake THERMOPHILES Yellowstone hot spring Pyrococcus furiosis In the 1960s biologists thought life would not tolerate temperatures anywhere near 80°C. But Thomas Brock found Thermus aquaticus that could live and reproduce near 100°C the temperature of boiling water. HOW HOT IS HOT? Pyrococcus furiosis was isolated from a geothermal sediment in Vulcano Island, Italy. Grows between 100-113°C with an optimum at 100°C. It has a generation time of 35 minutes. Produces enzymes which are extremely thermostable. It is the preferred source for Pfu DNA polymerase for PCR. HOW COLD IS COLD? Psychrophilic bacteria -20°C Cryobacterium roopkundense 0-15°C Cryobacterium psychrophilum 0-18°C ANTARCTIC MICROBIOLOGY ? 1. Where do they exist? 2. How do they survive? 3. Are they useful to us? ABOUT ANTARCTICA……..… Fifth largest continent 10% of earth’s Antarctica land surface 14 million sq. km. Iciest >98% ice coverage Driest - <10 cm rainfall Highest – Average 2.5 Km Windiest – >65 Knots/h Coldest – Vostock - 89°C POLYPHASIC TAXONOMY : PHENOTYPIC, CHEMOTAXONOMIC AND PHYLOGENETIC CHARACTERISTICS Soil, Sea water, Sea ice, Cell wall sugars Fast ice, Sediment Menaquinones Peptidoglycan Fatty acids Lipid profile G + C of DNA DNA-DNA hybridisation 16S rRNA gene sequence BACTERIA FROM ANTARCTICA GRAM NEGATIVE GRAM POSITIVE Psychrobacter salsus Planococcus maitriensis P. halophilus P. antarcticus Total bacteria P. adeliensis P. psychrophilus P. glaciei Kocuria polaris identified upto P. vallis Arthrobacter gangotriensis species level = P. aquaticus A. flavus P. salsus A. roseus ~ 1000 Pseudomonas antarctica A. kerguelensis P. meridiana A. antarcticus P. Proteolytica Leifsonia aurea Pseudonocardia antarctica L. rubra Sphingobacterium antarcticus Sporosarcina macmurdoensis Halomonas glaciei Exiguobacterium soli Marinomonas ushuaiensis ( 30/240) M. polaris Marinobacter maritimus UNIQUE FEATURES PSYCROPHILIC 2- 300C OLIGOTROPHIC COLD ACTIVE ENZYMES ANTIBIOTIC SENSITIVITY INCREASED UNSATURATED FATTY ACIDS IN HONOUR OF ….. Maitri Station Planococcus maitriensis McMurdo Pseudomonas antarctica Pseudonocardia antarctica Sphingobacterium antarcticus Arthrobacter antarcticus Sporosarcina macmurdoensis Planoccocus antarcticus Kerguelen Dakshin Gangotri Arthrobacter gangotriensis Arthrobacter kerguelensis E SAMPLE X T rRNA APPROACH R A C CELL ENRICHMENT T I O Antarctic N DNA C PCR L O N rDNA I N Soil G . Sea water . rDNA CLONES S E Sea ice SEQUENCING Q U rDNA SEQUENCES Fast ice E N C Sediment I rDNA DATABASE N G 16S rRNA GENE CLONES OF SEA WATER Phylogenetic neighbour Seawater Seawater + crude oil (120) (104) Roseobacter 28 (43%) 10 (35%) Sulfitobacter 11 (9%) 4 (4%) Staleya 1 1 Glaciecola 14 (12%) 9 (9%) Psychromonas 2 0 Colwellia 1 1 Marinomonas 2 2 Psychrobacter 0 6 Vibrio 1 0 Oleispira 0 1 Pseudomonas 1 0 Epsilonproteobactera 0 1 Arcobacter 0 1 Cytophaga – Flexibacter – Bacteriodetes 15 (13%) 20 (20%) Unidentified and Uncultured 40 (30%) 43 (43%) (Prabagaran et al., 2006, FEMS Microbial Ecol.; Shivaji et al., 2010, Res. Microbiol.) VERTICAL DISTRIBUTION OF BACTERIA IN A LAKE SEDIMENT FROM ANTARCTICA BY T-RFLP Subsurface (18-22 cm) Lake sediment core (136 cm) Subsurface (18-22 cm) Middle (60-64 cm) Middle (60-64 cm) Bottom (100-104cm) DNA- 2.5 g Bottom (100-104cm) Amplify 16S rRNA gene Digest with RsasI GeneScan Shivaji et al. Res microbiol, 2010 PRESUMPTIVE IDENTIFICATION OF TRFS GENERATED FOLLOWING RSAI DIGESTION OF 16S RRNA GENES FROM A LAKE SEDIMENT FROM ANTARCTICA TRF Species match by MiCA and confirmed by TRF Species match by MiCA and confirmed by (bp) in-silico analysis using Trifle/NEB (bp) in-silico analysis using Trifle/NEB 109 Rhodopseudomonas palustris strain GH 430 Alcaligenes faecalis subsp. faecalis strain 119 Janthinobacterium lividum strain GA01 437* Not identified 142 Alkaliphilus metalliredigens strain QYMF 440- Pseudomonas sp. lip23 147 Uncultured rumen bacterium strain F24-A02 442 208 Uncultured rumen bacterium strain 4C0d-12 440- Acinetobacter septicus AK001 0106 290 Paenibacillus illinoisensis strain 14 NRRL 442 NRS-1356T 453 Streptomyces sp. sd-45 301 Uncultured bacterium clone SJA-69 453 Cellulosimicrobium sp. TUT1222 309 Flavobacteriaceae bacterium strain CNU041 453 Cryobacterium sp. DR9 309 Pedobacter sp. strain DL3 457 Streptomyces armeniacus JCM 3070T 394* Not identified 457 Eubacterium lentum JCM 9979 400* Not identified 457 Brevibacterium celere KMM3637T 419 Polaromonas naphthalenivorans strain CJ2 457 Arthrobacter globiformis 168 DSM 20124T 427 Comamonas testosteroni RH 1104 467 Thermoanaerobacter sp. X514 427 Craurococcus roseus strain NS130 467 Tissierella praeacuta ATCC 25539T 427 Enterobacter pyrinus KCTC 2520T 474 Thermomonospora chromogena (Shivaji et al., 2010, Res. Microbiol.) 16S rRNA GENE CLONE LIBRARIES CONSTRUCTED USING A LAKE SEDIMENT FROM ANTARCTICA Phylum / Class 16S rRNA gene Library Subsurface Middle Bottom (18-22 cm) (60-64 cm) (100-104 cm) Number Library Number Library Number Library of (%) of (%) of (%) clones clones clones Proteobacteria Betaproteobacteria 9 6.8 0 0 0 0 Gammaproteobacteria 82 62.1 100 99.0 0 Bacteroidetes Flavobacteria 38 28.8 01 1.0 0 0 Actinobacteria Actinobacteria 2 1.5 0 0 3 4.7 Caldiserica Caldiserica 0 0 0 0 53 82.8 Firmicutes Bacilli 1 0.8 0 1 1.6 Clostridia 0 0 0 0 6 9.3 Unclassified 0 0 0 0 1 1.6 Shivaji et al. (2010) Res. Microbiol. ARE PSYCHROPHILES ENDEMIC TO ANTARCTICA ? Do similar species exist in Arctic? ARCTIC BACTERIA AS WORKHORSES OF BIOTECHNOLOGY : BIODIVERSITY OF BACTERIA AND BIOPROSPECTING FOR BIOMOLECULES LOVENBREEN GLACIER West Middle East SEDIMENT FROM KONGSFJORDEN NEW SPECIES FROM ARCTIC 1. Oceanisphaera arcticum - Kongsfjorden, Svalbard (2010) Int. J. Syst. Evol. Microbiol. 2. Cyclobacterium qasimii – Kongsfjorden, Svalbard (2010) Int. J. Syst. Evol. Microbiol. BACTERIAL DIVERSITY AND Kongsfjorden BIOPROSPECTING IN MIDTRE LOV’ENBREEN GLACIER, ARCTIC Bacterial abundance was greater at the convergence of the glacial melt water with the sea than at the snout. 117 bacterial strains were isolated from the sediments. Based on 16S rRNA gene sequence the isolates belonged to four phyla Actinobacteria, Bacilli, Flavobacteria and Proteobacteria. Isolates varied in growth temperature range (4-30°C), in tolerance to NaCl (0.1-1 M NaCl) and in growth pH range (2-13). Only 14 of 32 representative strains exhibited amylase, lipase and (or) protease activity and only one isolate (AsdM4-6) showed all three enzyme activities at 5 and 20 C respectively. More than half of the isolates were pigmented. Short-chain, unsaturated, branched, cyclic and cis fatty acids were predominant in the psychrophilic bacteria. Midtre Lov’enbreen Glacier (Reddy et al., 2009, Res. Microbiol.) BACTERIAL DIVERSITY AND BIOPROSPECTING IN KONGSFJORDEN AND NY-ALESUND, ARCTIC Bacterial abundance in marine sediments varied marginally. A total of 103 bacterial isolates based on the 16S rRNA gene sequence belonged to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria. The isolates varied in their growth temperature range (4–37ºC), in tolerance to NaCl (0.3–2 M NaCl) and growth pH range (2–11). 26 isolates exhibited amylase and lipase activity either at 5 or 20°C or at both the temperatures. A few of the representatives exhibited amylase and/or lipase activity only at 5ºC. None of the phylotypes exhibited protease activity. Most of the phylotypes were pigmented. Short chain, unsaturated, branched, cyclic and cis fatty acids were predominant in the Kongsfjorden and Ny-Alesund psychrophilic bacteria. (Srinivas et al., 2009, Curr. Microbiol) THE HIMALAYAN CONNECTION ROOPKUND GLACIER - 5029 m ROOPKUND LAKE Altitude : 4771 m Temperature : 2C / 6C Collection: September 2008 HAMTA GLACIER – 4270 m NEW SPECIES FROM HIMALAYAN GLACIERS 1. Paenibacillus glacialis - Kafni (2010) IJSEM. PINDARI GLACIER – 3627 m 2. Leifsonia kafniensis – Kafni (2009) IJSEM. 3. Leifsonia pindariensis - Pindari (2008) IJSEM. 4. Bacillus cecembensis – Pindari (2008) IJSEM. 5. Rhodotorula himalayensis – Roopkund (2008) Extremophiles. KAFNI GLACIER – 3853 m 6. Cryobacterium roopkundensis – Roopkund (2009) IJSEM. 7. Exiguobacterium indicum – Hamta (2007) IJSEM. 8. Planococcus stackebrandtii - Himalayas (2005) IJSEM. BACTERIA FROM HIMALAYAN GLACIERS Bacteria Roopkund Hamta Kafni Bacteria Roopkund Hamta Kafni Brevundimonas vesicularis + Bacillus subtilis + Pseudomonas mephitica + + B. arvi + B. globisporus + P. tremae + Dyadobacter fermentens + P. veronii + Pedobacter heparinus + P. borealis + + P. duraquae + P. migulae + Ewingella americana + P. cannabina + Pectobacterium carotovorum + P. trivialis + Bacillus acetylicum + P. corrugata + Variovorax paradoxus + P. jessenii + Serratia marcescens + P. mandeliii + Janthinobacter lividum + P. fluorescens + Arthrobacter boritolerans + P. frederiksbergensis + A. oxidans + + P. teessidea + A. stackebrandtii + P. poae + A. citreus + P. pavonaceae + A. psychrophenolicus + P. putida + A. polychromogenes + P. orientalis + A. sulfonivorans + P. brennerii + Brevibacterium antarcticum + P. Graminis + B. antiquum + Rhodococcus erythropolis + + Paenibacillus antarcticus
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