08/10/2005

Importance and Diversity of the Halophilic Archaea

Mădălin Enache

RIKEN, Bioresource Center, Japan Collection of Microorganisms

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

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.

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 :

Hypersaline environments & halophilic

microorganisms

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)

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)

Aerial view of a modern solar saltern facility in Grantsville, Utah (DasSarma 1995)

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

Physiological & biochemical properties

How they obtaining energy for growth?

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

Colonies of Halorubrumvacuolatum JCM 9060

Colonies of Halobacteriumsp. JCM 9447

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

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

Particularity of Halophilic Microorganisms

- lipids

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

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)

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

Particularity of Halophilic Microorganisms

- bacteriorhodopsine

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

How halophilic archaea cope with

salinity of extremely environment ?

• 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.

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

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

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

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

Some organic compatible solutes found in halophilicand halotolerant microorganisms

My research activity in Japan

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

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 + + + +

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

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

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

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

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

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

Formation of sulfide from sulfur

The paper impregnate with lead acetate became black color = positive result

Casein hydrolysis by halophilic archaea

Strain 22

Strain 20

Strain JCM 8864

Strain 21

Clear zone surrounding colony show a positive result

Utilization and acid production from glucides

BR2 BV2 BB8 TL6 GR2Arabinose +/- +/- -/- +/- -/-Raffinose +/- +/- +/- -/- +/-D-xylose +/- +/- -/- +/- -/-Maltose +/- +/- -/- +/- +/-Sucrose +/- -/- +/- +/- +/-Lactose -/- -/- -/- +/- +/-

Utilization / Acid production

+ = positive result

- = negative result

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

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