An excess of salt: lacusprofundi, an Antarctic (a model for exobiology) Shiladitya DasSarma Center of Marine Biotechnology University of Maryland Biotechnology Institute Baltimore, Maryland

Topics A psychrotrophic halophile: Halorubrum lacusprofundi Halophile diversity Hypersaline environments Mechanisms of adaptation Genomics and biology

1 Microbial growth characteristics

Nonhalophiles <0.2 M <1%

Slight 0.3-0.8 M 2-5 %

Moderate halophiles 0.8-2.5 M 5-15 %

Extreme halophiles 3-5 M 18-30 %

Obligate halophiles require high salt concentrations for growth

Psychrotroph Grows in cold temperatures (<10o C)

Distribution of hypersaline environments worldwide

•Tropical land/sea boundary •Permian halite deposits •Submarine brines salt diapir- associated •Temperate & arctic 13, GSL; 15, ; 9, Deep Lake

2 Deep Lake, Antarctica

™ Vestford Hills lake system (68 oS 78 oE) ™ Holocene glacial marine uplift (4,000 yrs old) ™ 0.64 km2 size, 36 m deep, monomictic ™ 28-32 % salinity (8-10 X sea salinity) ™ Thermally stratified (seasonal 10 oC to -20 oC) o o ™ Temp < 0 C for 8 months of year, 11.5 C Tmax . ™ Neutral pH, 360 mV redox, no H2S (Na2SO4 H20) Halorubrum lacusprofundi ™ Extremely low productivity (C-fix < 10g/m2/yr)

Halorubrum lacusprofundi • Isolated by Franzmann et al. 1988 • Dominant Deep Lake microbe • Grows in media containing (per L)

180 g NaCl, 75 g Mg2Cl, . 7.4 g KCl, 1.0 g CaCl2 H2O • Medium liquid to –20o C • Temperature minimum 2o C, optimum 31o C • size ~4.3 Mbp • GC composition 62 % • Mechanism of adaptation to psychrotropy?

3 Great Salt Lake Thalassic: 3900 sq. km area, 10 m deep; Halobacterium sp. NRC-1

Dead Sea Athalassic: 800 sq. km area, 340 m deep; Haloarcula marismortui

Great Salt Lake Dead Sea

4 Artificial hypersaline environments: solar salterns

Growth characteristics of halophiles

5 Ionic composition of Halobacterium cells

Intracellular concentrations: Ratios:

Na+ 1.37 +/- 0.21 M Cell Na+ /medium Na+ 0.30 K+ 4.57 +/- 0.12 M Cell K+ /medium K+ 143 Cl- 3.61 +/- 0.07 M Cell Na+ +K +/medium Na+ 1.32 Cell Cl- /medium Cl- 0.80

Tree of life

6 Diversity of Halophilic *

*Finished *Closure phase * *Shotgun phase * * *Low pass *

*

Adaptive features of hypersalinity (desiccation), high solar radiation, microaerobic conditions, temperature extremes, limited nutrients

Growth aerobic chemoorganotrophic facultative anaerobic phototrophic* Motility chemotaxis and phototaxis gas vesicle-mediated flotation* Cell envelope retinal pigments (BR, SRI & II, HR) S-layer sulfated glycoprotein archaeol (branched ether) lipids carotenoids* Enzymes salt requiring or tolerant* Genetics UV and radiation tolerant* genetic plasticity (IS elements)

7 Experimental tools Genetic selectable markers cloning vectors reporter genes expression vectors gene replacement methods

Genomic gene knockouts DNA arrays proteomics

Biophysical membrane protein analysis electronic diffraction methods X-ray structures

Halobacterium proteomics

2 5 3 7 pH gradient pH gradient

8 10.0% 4.2 9.0% Acidic proteins 8.0% 7.0% of Halobacterium 6.0% NRC-1 5.0% 4.0%

3.0%

2.0%

1.0%

0.0%

pI 4 4 .4 9 9 .9 4 4 4 .9 3. 3.9 4. 4.9 5 5.9 6.4 6. 7.4 7. 8.4 8 9.4 9.9 10. 10.9 11. 11.9 12. 12

Bsu Eco Mja Mth Sce Hal TFB (NRC-1 & Human) GyrA (NRC-1 & E. coli)

2-D crystalline lattice of BR in purple membrane

9 Complementarity of spectra of purple membrane and photosynthetic membrane

Radiation resistance of Halobacterium

UV Survival Curve For Halobacterium NRC1 (03/05/2002)

Dose (J/m^2) 0 20 70 100 150 200 250 300 350 400 1.00 Survival effect of halobacterium halobium strain NRC-1 on different doses of irradiation

10000000000 1000000000 100000000 10000000 1000000

100000 Series1 0.10 N/No 10000 N/No 1000 100

N um ber ofviable cells /m l 10 1

0 00 500 000 00 1 2000 4000 60 100 Irradiation dose (greys) 0.01 No, cfu before UV irradiation; N, cfu following UV irradiation

10 Halobacterium gas vesicles freeze-fracture, TEM, and cell flotation

ComparativeDifferential proteomic analysis of Halobacterium expression NRC-1 grown with under Light light and Dark (control).

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Control Light 10

8

6 Normal Volume

4

2

0 1 5 9 8 7 6 4 2 3 13 35 66 65 55 23 43 38 60 67 22 79 62 89 92 88 36 45 58 10 83 80 93 42 70 63 41 82 14 34 54 26 17 33 97 21 46 49 56 86 20 31 69 74 96 76 72 15 68 52 18 16 37 64 85 87 32 29 28 47 78 75 71 84 24 57 51 94 95 90 30 48 59 11 91 73 12 53 99 40 61 27 39 81 98 50 19 77 44 25 104 100 101 102 105 103 No. of Spots

11 Integrated view of the biology of Halobacterium

hν hν

OxlT LctP RbsAC UgpABCE H + BR HR SRI/HtrI SRII/HtrII (Ser) + formate H

Htr X (HemAT) Htr IV-VII oxalate lactate ribose glycerol Htr XIV-XVI 3-phosphate Htr IX, XII, XIII + CheAW HtrVIII spermidine ATP H ADP, P XVII, XVIII (?) PotABCD i putrescine Nutrient Uptake Energy Production Signal Transduction HtrIII (BasT)

CheY DppABCDF dipeptides sensor kinases

AppBCF oligopeptides response sensor regulators kinases

PutP proline chloride channel

Cat cationic 2- amino acids CysAT

amino acid amino acids 3- ABC transporter PstABC

Protein Translocation Anion Transport 3- 7S RNA SecDEFY SRP54 3- SRα SRP19 ArsAB ArsC Cation Transport 1- 1- K + K + H + Co 2+ Cu 2+ Fe 3+ Fe 3+ Zn 2+

Na + Cd 2+ Cd 2+ TrkAH KdpABC NhaC ZntX Cd efflux CbiNOQ NosFYiron HemUV ZurMA ATPase permease

Halorubrum lacusprofundi growth at reduced temperatures

12 Temperature regulated proteins

Summary • H. lacusprofundi is a novel psychrotrophic halophile • Halophiles are easily isolated and cultured • Halophiles have acidic proteins which are soluble and functional in hypersaline brine • Halophiles have novel features such as purple membrane, carotenoids, and gas vesicles • Halophiles are resistant to radiation and desiccation • Halophiles are excellent models for space/exobiology

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