importance and diversity of the halophilic archaea mălin ...riken, bioresource center, japan...
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08/10/2005
Importance and Diversity of the Halophilic Archaea
Mădălin Enache
RIKEN, Bioresource Center, Japan Collection of Microorganisms
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Introduction in Microbiology
Hypersaline environments & halophilic microorganisms
Physiological & biochemical properties
How they obtaining energy for growth?
Particularity of Halophilic Microorganisms
- lipids
- bacteriorhodopsine
How survive microorganisms to high salt concentration?
- salt-in strategy
-compatible solute strategy
Research activity in Japan
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Living organisms are composed of cells.
Plants and animals are called multicellular; they are composed of many cells and each cell depend on the other, cannot have an independent existence
Microorganisms are free-living cells. They are unicellular and able to carry out its life processes of growth, energy generation, and reproduction independently of other cells.
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Microorganisms – a microscopic organism consisting of a single cell.
Individually they can be see only by microscope
Microbiology – is the study of microorganisms
Microbiology study is important as:- basic biological science – understanding life process
- applied biological science – industrial process of biotechnology
Wide spread of biotechnology is based on diversity of microorganism which can be find in all three domain of life :
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Hypersaline environments & halophilic
microorganisms
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Categories of halophilic microorganisms
Category Growth salt range (M) Example
Halotolerant 0.0 - 5.2 Staphylococcus aureus
Slight halophile 0.2 - 0.5 Marine bacteria
Moderate halophile 0.5 - 2.5 Vibrio costicola
Borderline extreme halophile 1.5 - 4.0 Actinopolyspora halophila
Extreme halophile >2.5 Halobacterium, Halococcus
(After Ventosa A., 1989)
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Habitat of the halophilic archaea
Type of habitat Examples
Salterns Alicante, Spain
Solar salt Puerto Rico, Bonaire
High salt foods Salt fish
Salt mines Cheshire, UK
Salt lakes, neutral pH Great Salt Lake, UtahDead Sea, Israel
Salt lakes, alkaline pH Lake Magadi, KenyaOwens Lake, California
Salt lakes, cold Vestfold Hills, Antarctica
Salt ponds Widely distributed
(after Norton, C.F., 1992)
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Aerial view of a modern solar saltern facility in Grantsville, Utah (DasSarma 1995)
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The Salt Mountain in Slanic Prahova – Romania; The mountain cover partially the Lake Bride Cave, a habitat of new extremely halophilic archaea pertaining to Haloferax andHaloarcula genera
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Physiological & biochemical properties
How they obtaining energy for growth?
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Requires at least 1.5 M NaCl for growth
Aerobic or facultatively anaerobic
Mesophilic or thermotolerant
Neutrophilic or alkaliphilic
Insensitive to inhibitor antibiotic of Bacteria
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Colonies of Halorubrumvacuolatum JCM 9060
Colonies of Halobacteriumsp. JCM 9447
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Gram-stained cells of Natronococcusamylolyticus JCM 9655
Rod-shaped cells of Natronobacteirumnitratireduces JCM 10879
Triangular and square shaped cells of Haloarcula quadrata JCM 11048
Pleomorphic cells of Natrinema sp. XA3-1
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An integrated view of the biology of Halobacterium NRC-1
After Ng, Wailap Victor et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 22, 12176-12181
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Particularity of Halophilic Microorganisms
- lipids
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Esters
Esters are organic compounds formed by reaction between alcohol and acids. Esters formed from carboxylic acids have the general formula RCOOR.
Ethers
Ethers are organic compounds with formula R-O-R, where R is not equal to H. They may be derived from alcohols by elimination of water, but the major method is catalytic hydration of olefins.
R C O R R O R
O
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Structure of core lipidsO
O
RO
O
C20C20
C20C25
R
Structure of phospholipids
O
O
O
R
R
CH2
CH2
C
OP
O-
O
CH2
CH2OX
C OHHH
Phospholipid X
Phosphatidylglycerol (PG) HPhosphatidylglycerophosphate (PGP) -PO(OH)2Phosphatidylglycerosulfate (PGS) -SO2(OH)
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Structures of characteristic glycolipids of the halophilic archaea
R2-O-CH2
OR3 OH
HO
OH
HO-CH2
HO
O O
O
O O
CH2
Glycolipid R2 R3
DGD-1 (diglycosyl diether-1) H HTGD-1 (triglycosyl diether-1) β-galp HTGD-2 (triglycosyl diether-2) β-glcp HS-DGD-1 (sulfated diglycosyl diether-1) -SO2-OH HS-TGD-1 (sulfated triglycosyl diether-1) 3-SO3
--β-galp HS-TeGD (sulfated tetraglycosyl diether) 3-SO3
--β-galp α-galf
O
R CH
R CH2
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Particularity of Halophilic Microorganisms
- bacteriorhodopsine
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Model of the light mediated bacteriorhodopsin proton pump in the purple membrane of Halobacterium. The P stands for the protein to which the chromophore retinal is attached. Out an In designate oposite sides of the cytoplasmic membrane
ATPase
ATP
(Cisform)
Light
Purple membrane
Out
InADP + Pi
After Madigan M.T., Martinko J.M. and Parker J, 1997, pg. 748
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How halophilic archaea cope with
salinity of extremely environment ?
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• Living in media with high salt concentration poses a serious stress that halophiles have overcome through special processes or adaptations.
• The stress is represented by the microorganisms ability to maintain an internal ionic strength equals with their external environment.
• Osmosis is diffusion process in which molecules of water are transferred from an area of high concentration to an area of low concentration.
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Two fundamentally strategies exist within the microbial world that enable microorganisms to cope with stress generated from high salt environments:
1.The salt-in strategy
2.The compatible-solute strategy
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The salt-in strategy
Is used by two phylogenetically unrelated groups:
1. Halobacteriales – aerobic extremely halophilic archaea
2. Haloanaerobiales – anaerobic halophilic bacteria
• Intracellular ionic concentration are similar to those of
surrounding medium
• Intracellular ionic composition are different to those of
surrounding medium
• Enzyme and structural cell components are adapted to the
presence of high salt concentration
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An integrated view of the biology of Halobacterium NRC-1
After Ng, Wailap Victor et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 22, 12176-12181
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The compatible-solute strategy
Is used by:
1. Nonhalophilic and halotolerant microorganisms
2. Slight halophilic and moderately halophilic bacteria
• Intracellular ionic concentration are different to those of
surrounding medium
• Osmotic pressure of the medium is balanced by organic
compatible solutes
• Enzyme and structural cell components no need special
adaptation for their activity
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Some organic compatible solutes found in halophilicand halotolerant microorganisms
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My research activity in Japan
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Research activity in Romania & Japan
Some chemical properties of investigated lakes
Main ions (g/l) No. of strains
Cl- Na+ Mg2+ Isolated total Archaea Bacteria
1 Red Bath 7.9 74.9 44 0.05 17 2 15 BR 2
2 Shepherd Bath 8.7 97.4 44 0.03 48 8 40 BB8
3 Green Bath 9.0 138.5 71 0.05 32 5 27 BV2
4 Telega 8.3 161 - - 20 20 - TL6
5 Bride Cave 8.3 254.6 113 0.06 42 10 32 GR2
Tested strainsNo Lake pH
value
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G + C content and lipids profile of investigated strains
Mol% G+C PG PGP DGA-1 S-DGA-1
BR2 65.8 + + + +
BV2 63.6 + + - +
BB8 63.8 + + + +
TL6 63.7 + + - +
GR2 64.9 + + + +
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TLC of membrane lipids of investigated strains
PG
BR2 BV2 BB8 TL6 GR2
S-DGA-1
DGA-1
PGP-Me
BR2 BV2 BB8 TL6 GR2
Phospholipids patterns Glycolipids patterns
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Growth requirements for investigated strains
BR2 BV2 BB8 TL6 GR2
Range of NaCl conc. (M) for growth 2.0-5.5 2.0-5.5 1.5-5.5 1.0-5.5 1.0-5.5
Optimum NaCl conc. (M) for growth 3.0-3.5 3.0-3.5 3.0-3.5 2.5-3.5 3.0
Range of MgCl2 conc. (M) for growth 0-1 0-1 0-1 0-1 0-1
Optimum of MgCl2 conc. (M) for growth 0.4 0.4 0.4 0.4 0.4
Range of temperature (0C) for growth 15-56 16-51 18-51 23-51 20-48
Optimum temperature for (0C) growth 36-41 36-41 38 38-48 36-41
Range of pH values for growth 5.0-9.0 5.0-8.5 5.0-9.0 6.0-8.5 5.0-9.0
Optimum pH values for growth 6.5-7.0 7.0 6.0-7.0 7.0-7.5 6.5-7.5
Anaerobic growth in presence of nitrate negative negative negative negative negative
Anaerobic growth in presence of Arg and DMSO negative negative negative negative negative
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Effect of NaCl concentration on the growth and pigmentation on halophilic archaea Haloferax sp.
0.0 0.1 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 NaCl - in molar concentration (M); i.e. 1M = 58.5 g of NaCl dissolved in one litter of distill water
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Effect of temperature on the growth and pigmentation on halophilic archaea Haloferax sp.
61 56 51 48 45 41 38 36 34 31
Temperature in degrees centigrade
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Halogeometricum borinquense ATCC 700274 (AF002984)
Haloferax sulfurifontis JCM 12327 (AY458601)
Haloferax mediterranei ATCC 33500 (D11107)
BV2
TL6
BR2
GR2
BB8
Haloferax gibbonsii ATCC 33959 (D13378)
Haloferax lucentense JCM 9276 (AH003665)
Haloferax volcanii ATCC 29605 (K00421)
Haloferax alexandrinus JCM 10717 (AB037474)
Haloferax denitrificans ATCC 35960 (D14128)
0.005
Phylogenetic tree showing the position of investigated strains. The tree was reconstructed by the neighbor-joining method derived from sequence of 16S rDNA.
Haloferax denitrificans
Haloferax alexandrinus
Haloferax volcanii
Haloferax lucentense
Haloferax gibonsii
BB8
GR2
BR2
TL6
BV2
Haloferax mediterranei
Haloferax sulfurifontis
Halogeometricum borinquense
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Biochemical test
reduction of nitrate: all investigated strains are negative
formation of sulfide from thiosulphate: all investigated strains are positive
formation of indol: all investigated strains are positive
catalase and oxidase activity: all investigated strains are positive
hydrolysis of starch: all investigated strains are positive
hydrolysis of tween 80: all investigated strains are positive
hydrolysis of gelatin: all investigated strains are negative
hydrolysis of casein: all investigated strains are negative
Sensitivity to antibioticsall investigated strains are sensitive to: novobiocin, anysomicin, aphidicolin
and rifampicin
all investigated strains are insensitive to: bacitracin, erythromycin, penicillin, ampicilin, chloramphenicol and neomycin
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Formation of sulfide from sulfur
The paper impregnate with lead acetate became black color = positive result
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Casein hydrolysis by halophilic archaea
Strain 22
Strain 20
Strain JCM 8864
Strain 21
Clear zone surrounding colony show a positive result
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Utilization and acid production from glucides
BR2 BV2 BB8 TL6 GR2Arabinose +/- +/- -/- +/- -/-Raffinose +/- +/- +/- -/- +/-D-xylose +/- +/- -/- +/- -/-Maltose +/- +/- -/- +/- +/-Sucrose +/- -/- +/- +/- +/-Lactose -/- -/- -/- +/- +/-
Utilization / Acid production
+ = positive result
- = negative result
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CONCLUSION
• Halophilic archaea are salt loving microorganisms which live in special environment where another living organisms cannot survive
• They have unique lipid in cell membrane, ether bondage which differ from ester bondage of other living organisms
• Bacteriorhodopsine is a unique protein which can transform solar energy in chemical energy
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Warmest thanks for:- Japan Society for the Promotion of Science
- Dr. Takashi Itoh – RIKEN, Japan Collection of
Microorganisms
- Dr. Masahiro Kamekura – Noda Institute for
Scientific Research