COLD-LOVING 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 : 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 level = P. aquaticus A. flavus P. salsus A. roseus ~ 1000 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 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 Betaproteobacteria 9 6.8 0 0 0 0 82 62.1 100 99.0 0 Bacteroidetes Flavobacteria 38 28.8 01 1.0 0 0 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 : 2C / 6C 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 + Okibacterium fritillariae + P. Macquariensis + Metagenomic libraries of Roopkund, Pindari and Hamta glaciers (2500 clones)

Gem, Gemmatimonadetes Pro, Proteobacteria Len, Lentosphaerae Aci, Acidobacteria Nit, Nitrospirae Act, Actinobacteria OP11, candidate divisions OP11; CFB, Cytophaga–Flavo–Bacteroides Pla, Planctomycetes Chf, Chloroflexi Spi, Spirochaetae Cya, Ten, Tenericutes De-Th, Deinococcus and TM7, TM7_s TM7a (candidate Themomicrobia Elu,Elusimicrobia phylum) Unc, unculturables Fir, Firmicutes Ver, Verrucomicrobia.

Samoylov Island, Siberia. Siberian tundra Schirmacher Oasis, Antarctica Bench Glacier, Alaska John Evans Glacier, Canada Mount Everest

Pindari Glacier, Himalayas Roopkund Glacier, Himalayas Kafni glacier, Himalayas

Extremophiles (2010) 14 : 377-395 Extremophiles(2010)15:1-22 Extremophiles (2011) SIMILARITIES OF BACTERIA FROM ANTARCTICA AND HIMALAYAS

 Unique to each glacier  Psychrophilic  Oligotrophic  Produce cold active enzymes ANTARCTIC MICROBIOLOGY ? 1. Where do they exist? 2. How do they survive? 3. Are they useful to us? PSYCHROPHILC BACTERIA : HOW DO THEY SURVIVE?

1. Ability to sense temperature.

2. Ability to modulate membrane composition.

3. Ability to undergo transcription and translation at low temperature.

4. Ability to catalyse reactions at low temperature.

5. Presence of genes required for growth at low temperature.

250C 50C TWO-COMPONENT SIGNAL TRANSDUCTION PATHWAY FOR THE PERCEPTION AND TRANSDUCTION OF LOW TEMPERATURE IN BACTERIA

ORGANIZATION OF SENSOR AND RESPONSE REGULATOR IN SYNECHOCYSTIS PCC6803

(i) The sensor (S) perceives the signal due to rigidification of the membrane, gets activated by phosphorylation (Sa) and transfers the phosphate to the response regulator (R). (ii) The phosphorylated receptor (Ra) is a transcription regulator and activates cold-inducible genes whose products would help in cold adaptation. (iii) The model also demonstrates a link between expression of cold- inducible genes and DNA supercoiling.

(i) The sensor has two transmembrane domains (shaded cylinders). (ii) The HAMP domain (histidine – kinase – adenylyl – cyclase – methyl – binding protein phosphatase). (iii) The PAS (PER - ARNF – SIM) domain. (iv) The histidine kinase domain and the histidine residue (vertical rectangle H). (v) Response regulator has a receiver domain with an aspartate residue (D), the transcriptional activation domain (ARNT) and the DNA-binding domain (HMG). CHANGES IN MEMBRANE FLUIDITY

0 0 Changes in fatty 25 C 5 C acid composition

Role of desaturases Interaction of carotenoids Role of cti Shivaji, and Prakash (2010) Arch. Microbiol. 192 : 85-95. TEMPERATURE-DEPENDENT SYNTHESIS OF CAROTENOIDS OF SPHINGOBACTERIUM ANTARCTICUS

Pigment Retention time (min) Relative Quantity (%) (mean + S.D.) 5oC 25oC POLAR PS1 Unidentified 3.76 6.12 + 2.92 3.69 + 1.75 PS2 Zeaxanthin 4.38 79.23 + 3.63 49.28 + 2.45 LESS POLAR PS3 b-Cryptoxanthin 8.86 12.84 + 1.88 30.19 + 0.82 PS4 Unidentified 10.08 0.61 + 0.22 1.78 + 0.53 PS5 b-Carotene 24.00 1.71 + 0.20 15.04 + 2.80

250C 50C

Jagannadham et al. J. Bacteriol. (1991); Arch. Microbiol. (2000) CHANGES IN MEMBRANE FLUIDITY

Changes in fatty 250C 50C acid composition

Role of desaturases Role of cti Interaction of carotenoids

Shivaji and Prakash (2010) Arch. Microbiol. 192 : 85-95. COLD ADAPTATION : ACYL-LIPID DESATURASES CYANOBACTERIA OF CYANOBACTERIA

Optimal Shift from growth Low growth 35 to 25°C temperature temperature D9 Desaturase C C18:0 C18:1 (9)

Mesophilic Cyanobacteria D12 Desaturase A

C18:1 C18:2(9,12)

desaturases D15 Desaturase B Saturated FAs Unsaturated FAs C18:2 C18:3(9,12,15)

D6 Desaturase D  Acyl lipid desaturases [Cyanobacteria and Plants] C18:3 C18:4(6,9,12,15)  Acyl-CoA desaturases [Animals, Yeast and fungi]  Acyl-ACP desaturases [Plant plastids]

Shivaji and Prakash (2010) Arch. Microbiol. FATTY ACIDS AND COLD ADAPTATION

OPTIMUM TEMP : 35˚C MESOPHILIC CYANOBACTERIA COLD STRESS : 22˚C

ANTARCTIC CYANOBACTERIA COLD ADAPTED 5˚C - 30˚C  ARE THE DESATURASES DIFFERENT ?  ARE THE DESATURASES COLD INDUCIBLE ?

FATTY ACID COMPOSITION OF PSYCHROPHILIC NOSTOC FROM ANTARCTICA Fatty acid mole % Fatty acid Heterocyst 90C 250C

C16:0 12.0 13.3 C16:1 (9) 27.7 24.6

C18:0 1.4 1.4 Heterocyst

C18:1 (9) 5.3 13.4 Decrease in 18:1 is indicative of desA activity C18:1 (11) 2.9 3.3 C18:2 (9,12) 16.0 28.3 Increase in 18:3 is indicative of desB activity

C18:3 (9,12,15) 34.6 15.4 (Chintalapati et al., 2006, Biochem. J.) ORF of the desaturase gene cluster in Nostoc desC desA desB

822bp 1053bp 1077bp RT-PCR for des genes in Nostoc

Minutes 0 15 30 45 60 120 150 desA

desB

desC

(Chintalapati et al., 2006, Biochem. J.) Biochemical Journal (2006) 319 : 207-214

CONCLUSIONS 1. A new desaturase gene, desC2 gene, functional at 9 position of the fatty acids has been discovered. 2. Unlike mesophilic des A and B which are induced at low temperature, desA, desB, desC and desC2 are constitutively expressed in Antarctic Nostoc. SCREENING FOR GENES REQUIRED FOR GROWTH AT LOW TEMPERATURE

Pseudomonas syringae

40C 220C 280C TRANSPOSON MUTAGENESIS

No growth Retarded growth

40C 220C 280C Cold Sensitive Mutant GROWTH OF Pseudomonas syringae AND CSM1 4 4 5 4 4 3 3 5 5 4 ° ° 28°C 4 C 3 22 C 3 1 2 2 2 1 2 1 2

3.5 4˚C 3.5 22˚C 3.5 28˚C 3 3 3 2.5 2.5 2.5

2 2 2

600

600 600 1.5 OD 1.5

1.5 OD OD 1 1 1 0.5 0.5 0.5 0 0 0 1 3 5 7 9 11 13 15 17 19 21 0 12 20 28 36 44 52 60 68 76 84 92 102 1 3 5 7 9 11 13 15 17 19 Time (h) Time (h) Time (h) 4 5 1 = P. syringae; 2 = P. syringae with pGL10; 3 3 = Complemented2 CSM1; 4 = CSM1; 5= CSM1 with pGL10 1 30 out of 3000 mutants showed delayed growth at 4˚C (Singh et al. Appl. Environ.Microbiol 2009) ANALYSIS OF CSM1

 CSM1 has only one transposon insertion in its genome.  Sequencing of the flanking regions indicated that the mutated gene is trmE.  Blast analysis indicated maximum similarity with trmE of : Halomonas cupida Brevibacterium linens Bacillus megaterium Pseudomonas fluorescens

t-RNA MODIFICATION GTPase (trmE)

 trmE exhibits GTPase activity.  trmE modifies a uridine (U34) in tRNA at the wobbel position to 5- methylaminomethyl-uridine.  The modified U34 serves as a recognition nucleotide during codon- anticodon interaction.  Mutants in trmE lead to frameshift mutation during translation. EXPRESSION OF trmE OF P. syringae 22C 22C – 4C 22C - 22C 22C - 28C

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 bp 800 A 500 528 bp

B 23S 16S 5S

0.75 C

0.5

0.25

transcript/ total RNA trmE

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (min)

 Lane 1 culture grown at 22°C  Lane 2-6 culture grown at 22°C and shifted to 4°C for 10, 20, 30, 45 and 60 min  Lane 7-11 culture grown at 22°C without shift after 10, 20, 30, 45 and 60 min  Lane 12-16 culture grown at 22°C and shifted to 28°C for 10, 20, 30, 45 and 60 min (Singh et al. Appl. Environ.Microbiol 2009) DELETION ANALYSIS OF trmE-PROMOTER

j) Deletion of -100 to +400. 60 50 -) 50 40 40 30 30 20 20

10 10 -gal activity galactosidase galactosidase activity

galactosidase activity galactosidase 0 0 a b c d e f g h I j a b c d e f g h I j Promoter construct Promoter construct Ash57 AGCTACAAGTCGATGGCCCGCATGCGCGCTGTGGCACCAAAACTGGCCGCTCTGAAAGAGCAGTTCGGT

GATGATCGCCAGAAAATGTCGCAAGCGATGATGGAGCTGTACAAGAAAGAGAAGATCAATCCGCTGGGC trmE

GGCTGCTTGCCGATTCTGGTGCAGATGCCGGTCTTCCTGTCCTTGTACTGGGTACTTCTGGAAAGTGT UP element PROMOTER TGAAATGCGCCAGGCGCCGTTCATGCTCTGGATTACCGACCTGTCGATCAAGGACCCGTTCTTCATTCT -35 -10 +1 GCCGATCATCATGGGCGCAACCATGTTCATCCAGCAGCGTCTGAACCCGACTCCTCCGGATCCGATGCA OF P. syringae

GGCCAAGGTGATGAAGCTGATGCCAATCATCTTCACCTTCTTCTTCCTGTGGTTCCCGGCTGGTCTGGT

GCTGTACTGGGTTGTGAACAACTGCCTGTCCATCGCCCAACAGTGGTACATCACACGTAAGGTCGAAGC cold box DEAD box TGCTACCAAAAAAGCAGCCGAGTAACTTACTCTGGTGAGCACCACTCAAAACGCCCCCTAGTGGGGCGT Conserved region TTTGCTTTCCATCACTTTTGTTTTGAGGCCTGTTTTATG------Ash59 SDS EXPRESSION OF trmE OF P. syringae 22C 22C – 4C 22C - 22C 22C - 28C M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  Lane 1 culture grown at 22°C  Lane 2-6 culture grown at 22°C and shifted

M 1 2 3 4 5 6 7 8 9 10 11 13 15 16 to 4°C for 10, 20, 30, 45 and 60 min  Lane 7-11 culture grown at 22°C without shift after 10, 20, 30, 45 and 60 min 1

0.75  Lane 12-16 culture grown at 22°C and

transcript/ total 0.5 RNA shifted to 28°C for 10, 20, 30, 45 and 60 min

0.25 galactosidase

- 0 β 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (min) (Singh et al. Appl. Environ.Microbiol 2009) CLONING AND PURIFICATION OF TrmE FROM Pseusdomonas syringae 1. GTPase was over expressed in pET28 b 2. Over expressed protein has (N-terminal)" His tag” 3. Purified by Ni-NTA agarose column

M U 1 2 3 4 kDa M 1 2 3 4 5 6 97 66 53 kDa 45

30

PROPERTIES OF t-RNA MODIFICATION GTPase 1. Mol. Wt is 49 kDa. Not inhibited by Levamisole. K and Mg required. 2. Exhibits optimum activity 12 - 15°C and 30% of the activity at 0°C. 3. Hydrolyses dGTP and GTP, but not dGDP, ATP, dATP, CTP, dCTP, UTP and dTTP. 4. Irreversibly inactivated at 70°C. (Singh and Shivaji (2010) Res. Microbiol. 161 : 46-50). GROWTH OF Pseudomonas syringae AND CSM1

3.5 3 4°C 2.5

2 600 OD 1.5 CONCLUSIONS 1

0.5 0 In Pseudomonas syringae (Lz4W) 0 12 20 28 36 44 52 60 68 76 84 92 102 Time (h) trmE is a cold inducible gene 3.5 trmE is transcriptionally regulated 3 22°C 2.5 2 trmE has a cold inducible promoter 1.5600 OD 1 and 0.5 0 trmE is required for growth at 1 3 5 7 9 11 13 15 17 19 Time (h) low temperature. 3.5 WT (■) 3 28°C 2.5 WT with pGL10(Δ) 2 1.5600 OD 1 CSM1(▲) 0.5 0 Complemented CSM1(◊) (Singh et al., 2009, 1 3 5 7 9 11 13 15 17 19 21 Time (h) CSM1 with pGL10 (♦) Appl. Environ. Microbiol) GROWTH OF CSM2 AAT SEQUENCE

Sundareswaran Arch. Microbiol. (2010) CANDIDATE GENES INVOLVED IN COLD ADAPTATION

Gene Description Reference rbfA ribosome-associated factor Bing et al. (2003) csdA DEAD-box helicase Lim et al. (2000) CspA Cold shock proteins Thieringer et al. (1998) pnp polynucleotide phosphorylase Luttinger et al. (1996) hns DNA-binding histone-like nucleoid protein Dersch et al. (1994) bipA ribosome-associated GTPase Pfennig & Flower (2001) oppA Oligopeptide binding protein Borezee et al. (2000) recD* a DNA recombination and repair gene Regha et al. (2005) hutU* histidine utilization operon Kamala et al. (2002) trmE* tRNA modification GTPase Singh & Shivaji (2010) AAT* Aspartate amino transferase Sundareswaran et al. (2010)

*All these studies were done using a psychrophilic bacterium. The other studies were done using mesophilic bacteria ANTARCTIC MICROBIOLOGY ? 1. Where do they exist? 2. How do they survive? 3. Are they useful to us? BIOTECHNOLOGICAL POTENTIAL : COLD ACTIVE ENZYMES

ENZYME ORGANISM APPLICATIONS Metalloprotease Sphingomonas paucimobilis Food, detergents, Serine peptidase PA-43 subarctic bacterium Food, detergents, Lipase Aspergillus nidulans WG 312 Food, detergents, cosmetics Alkaline phosphatase Vibrio sp. G15-21 Molecular biology Triose phosphate Isomerase Vibrio marinus Biotransformation Chitinase A Arthrobacter sp. TAD 20 Food, health products Cellulase Fibrobacter succinogenes Animal feed, textiles, d Pectate lyase Pseudoalteromonas halplanktis Cheese ripening and wine Xylanase Cryptococcus adeliae Fermentation -Amylase Alteromonas haloplanktis Detergents, fermentation -Lactamase Psychrobacter immobilis Antibiotic degradation Catalase Vibrio rumoensis Water treatment Malate synthase Colwellia maris Biotransformation Carotenoid pigments Micrococcus roseus Dietary supplement, Sphingobacterium antarcticus Aantioxidants RNA polymerase / RNAase Pseudomonas sp. Molecular Biology ENZYMES OF BIOTECHNOLOGICAL POTENTIAL

Protease RNAase Alkaline RNA t-RNA Phosphatase Polymerase modification C. humicola P. fluorescens S. antarcticus P. syringae GTPase

Opt. Temperature(°C) 37 37 37 37 12 -18

Activity at 5°C (%) 15 – 20 15 – 20 15 – 20 10 – 15 65

Inactivated (°C) 56 65 65 37 70

(min) 10 20 20 15 30

Freeze thaw (cycles) R, 20 R, 20 R, 20 - -

Reddy et al. (1994) FEMS Microbiol. Letts.; Uma et al. (1999) FEBS Lett.; Alam et al. (2005) Enz. Microb.Technol.; Singh et al. (2010) Res. Microbiol. INFLUENCE OF TEMPERATURE ON ANTAGONISTIC PROPERTIES OF ISOLATES FROM ARCTIC

Activity at 18°C > 4°C

Activity at 4°C > 18°C

The differential inhibitory extent between selected producers (spot) and sensitive isolates (lawn) at 4 and 18 °C respectively is shown SCHEMATIC INTERACTION BETWEEN VARIOUS ISOLATES DEPENDING ON THEIR PSR STATUS

Nearest Phylogenetic Neighbour* Number of interactive types PRS PR PS SR P S Arthrobacter. alpinus S6-3(T) 1 - - - - - A. oxydans DSM 20119(T) 1 1 1 3 - 1 A. phenanthrenivorans Sphe3(T) 1 - - 2 - - A. polychromogenes DSM 20136(T) - 1 - 3 - - A. psychrochitiniphilus GP3(T) - 4 - 1 - - A. scleromae YH-2001(T) 1 - - 1 - - A. sulfonivorans ALL(T) 1 1 - - - - Flavobacter. limicola ST-82(T) - 1 - 4 - - Pseudomonas antarcticus LMG 22078(T) - - - 3 - - P. mandelii CIP 105273(T) - 1 - - 1 10 P. frederiksbergensis JAJ28(T) 1 3 - - - 6 11 6 12 1 17 1 17

K25 is a PSR interactive type in relation to K1, K2, K3 and K6. The dotted triangle is the probable antagonistic network with K25 as P, K3 as S and K1 as R.

The P–S, S–R and R–P relationship between K25, K3 and K1 is shown: K2 is a producer and K25 is sensitive K6 is a producer and K25 is resistant (Prasad et al., 2011, FEMS) CONCLUSIONS 1. A number of new genera and species of bacteria have been identified from cold habitats in Antarctica, Arctic, Himalayan glaciers, deep sea and stratosphere. 2. The rRNA approach revealed a greater heterogeneity of bacterial genera in the above habitats. 3. A number of cold-active enzymes with potential in biotech industry have been identified. 4. Cold-sensitive mutants of psychrophilic Pseudomonas syringae were generated by transposon mutagenesis. 5. AAT (coding for Aspartate aminotransferase) and t-RNA modification GTPase (trmE) have been identified as genes required for cold adaptation 6. Both AAT and trmE are cold active enzymes. 7. Complementation of the mutated trmE in CSM1 and AAT in CSM2 transformed the mutants from a cold-sensitive phenotype to a cold- resistant phenotype like the wild type cells. “The micro-organism is always right, your friend and a sensitive partner………”

“If you take care of your microbial friends they will take care of your future”

Dr. D. Perlman ACKNOWLEDGEMENTS Dr M K Chattopadhyay Dr J S S Prakash Dr K Suresh Dr Pavan K Pindi Dr Bhaskar Bhadra Dr S R Prabagaran Dr Pratima Gupta Dr Smita Dube Mr G S N Reddy Mrs Zareena Begum Mr V R Sundareswaran Mr M D Kiran Mr Suresh K Chintalapati Mr Ashish Kumar Singh Ms Preeti Chaturvedi Mr V Prabahar Ms Ruth Manorama Dr Anil Kumar Rs:…….. DBT , DST, JSPS, IFCPAR, NCAOR Dr T N R Srinivas