Thermophilic and halophilic extremophiles

265

Thermophilic Michael

T Madigan*

The microbiology moving field with isolation molecular

and halophilic

biomolecules. The on our understanding

structural

biochemistry.

iDivisIon

of MIcrobiology, USA; e-mall:

of Microbial

Sciences, and Biogeochemistry, 91904, Israel;

results of these of prokaryotic

Southern Illinois [email protected]

and Molecular

Ecology,

The Moshe Shllo Minerva The Hebrew University e-mail: [email protected]

Opinion

Oren?

of extremely hot or saline habitats is a fast many new successes in the enrichment and

halophilic new light

Current

and Aharon

of new organisms and in an understanding factors that impart stability on thermostable

Addresses *Department IL 62901-6508,

extremophiles

in Microbiology

of

Although growth

and

studies have shed diversity and

Unlverslty,

Carbondale,

The lnstltute

of Life

Science

Ltd

ISSN

rcccntly been recognized. of ‘I’homas Brock in the

only \vork [S]).

Karl have

Stetter isolated

of hl’perthcrmophiles is likely to be

gmc

most

extreme

temperau~rc hydrothermal

optima vents

ride

is ins id

of knonn

hyperrhermophiltts,

those

ubovc [6** .7’].

I’,~l~/clhis,filfI1Nl’ii

[ 1O’j

mophiles at high

and hyperthcrmophil~s) 121 and those salt concentrations (halophilcs) [A].

IntcrcsCngly environmental growth.

I:or

cussed in less dards their

example,

in this review rhan 3 hl i’La(:l cxtrcmophilcs popularity;

appeal

as models

unusual applied

Table

cxtremophilcs extreme,

in

do but some of

just

tolerate

unable (‘l:,lble l)!

their

to grow below 90°C: or an> biologist’s stan-

13~

are thus fascinatiry oyanisms. reccnr years is 3 testament to life

forms

uscfuI

in

and both

as

~Whzmp,~trr.r

kntdhi

;uxeptors. for any

tcnlpcTaturcs

(lzl’c:)

autoclnve

I! ,fhw//.N has knoun organism

the

(and respechighcst (‘lhhle 1)

occurs up to 113’(:, this wmpcraturw cultures of ;I one hour trcutment in the

[lo*].

gro\v

actiiall) require it for the organisms to bc dis-

arc

of primitive

hiomolecules research.

not

rhat

and

of these prokaryotcs are cheniolithotrophic tIpxidizcrs, reducing NOor (:Oz,

as electron

with

IOOY~, inhabit submarine ‘IWO examples of srlch organ-

[IO’]. Although growth of I:‘,fN?wli;i organism can survi1.e c\cn higher /?fi//w/.N remain viable following

of these remarkable uzmpcraturc (ther-

Howcviceberg. as

that man?; more [Y] exist.

\~ould salinit);

on t\vo groups grow at high

the 1070s

has rev-e&d [Xl and Bacteria

tively cardin31

rc\aiccv foc~~ses those that

Following 1960s and

probing Archaea

\Virhin the past 30 years a grcac diversity of microorganisms has been discovcrcd living under conditions that humans

and this organisms.

prokqor abovc

80°C

(‘l:dkle 1) [4.6”,7’]. the tip of the

only

Introduction

high [l]

-

with been

and colleagues at Kegensover 30 gcncra and 70

[I 11. 120th autotrophic)

c:~ll cxtrcnlc: high or low tcmper3tllrc or pH, or high prcssurc. ‘Iliac arc the ‘estrcniophiles’

(organisms 4.580°C:) have

[4] have pioneering

‘1%~

1369-5274

prokaryotes between

optima

HO years. hYpcrthermophiles temperature optima of

environmental hyperthcrlnophilic

2:265-269

prokaryotes

known for over otes with growth

species cr, this

http://biomednet.com/elecref/1369527400200265 c Elsevier

thermophilic temperature

(reviewcd in burg. Germany

Center for Marine of Jerusalem, Jerusalem

1999,

ThermophilWhyperthermophilic and their habitats

and their

s011rccs of basic

and

‘I‘he mcthanogen .I~~~~NNo~J~~~Ls k~~w’/fv~ grows at temperatures up to 1 lO”<: Ill]. .l/c’t/l~~t~o/~~~u.c was isolat4 from the walls of ‘black smoker’ hydrothermal vent chimneys who-c it reaches (I-1 ,fil/tu//ii ITsing

n~lmbers of 10” pt‘r grim was also isolated from

nucleic

prokatytcs, cd in chimnq thermal XO”(:

acid both

grdients across the

prohcs.

Archaca walls [12],

of chimnq chimney

several

morphological

and l’wxcria, suggesting

(wmperaturcs 0.5 cm thick

can

the

most

[(I”]

[ lO’]). rypcs

have been that

these

wry

b);

wall of the chimnq)

many diffcrcnt

may contain ing perhaps

rock

walls

more: than [6”,12]

microbial populations, thcrmophilic of all lift forms.

includ-

1

The most Group/Genus

extreme

hyperthermophilic

and species

Hyperthermophiles Aquifex pyrophilus Pyrolobus fumarii Halophiles Actinopolyspora halophila Halobacterium salinarum

and

Phylogenetlc

Bacteria Archaea

Bacteria Archaea

halophilic

prokaryotes.

domain

Growth

Reference

condltlons

Minimum

Optimum

MaxImum

67’C 9o”c

85°C 106°C

95°C 113V

2 M NaCl 3 M NaCl

2.6-3.4 4-5

M NaCl M NaCl

Saturation Saturation

(5.2 M NaCI) (5.2 M NaCI)

of

dercctcompact

[7-l

IlO’1 [531 [541

266

Ecology

and industrial

microbiology

In a phylogenetic sense, species of hyperthermophiles typically reside on rather short branches that emerge near the root of their respective domain [6”,7’,13]. The ‘Korarchaeota’, a newly recognized kingdom of Archaea, also represent an early branching group of hyperthermophiles [8]. This finding suggests that hyperthermophiles are the least evolved of extant organisms and thus likely share more features with early life forms than do any other extant prokaryotes. This hypothesis is also consistent with the widely held [2,6”] but not universally held [2,14] view that life first evolved on a hot planet Earth. The genomes of several hyperthermophiles have been sequenced, including those of Methanococcus jannaschii [ 151 and Aquzjkx aeolicus [ 161.The genomeof M.jannaschi wasthe first archaealgenome to be completely sequencedand has confirmed the phylogenetic stature of the Archaea; over 50% of the genesof M.jannaschii have no counterparts in Bacteria or Eukarya [15]. Analyses of the M. jannaschi genome sequence have also revealed several unsuspected genetic capacities,such asgenesencoding a functional ribulose bisphosphate carboxylase/oxygenase (RubisCO) [17”]. The genus Aguife contains the most thermophilic of all known Bacteria (Table 1); predictably, it is alsothe earliest branching organismon the phylogenetic tree of Bacteria [6”,7’]. The relatively smallgenome of A. aeokus (1.5 Mb, only one third the size of that of Escheri&a co/i [16]) is the smallest genome found to date in an autotrophic organism[ 181.

Molecular

adaptations

kandleri and Pyrodictium occulturn, two prokaryotes capableof growth up to 110°C.Chaperoninslike the thermosomefunction to bind heat denatured proteins, prevent their aggregation,and refold them into their active form [24].

Several factors may combine to afford heat stability to DNA in hyperthermophiles including high levels of K+ [ZO], reverse DNA gyrase [25’,26,27] and histone or other DNA-binding proteins [28”,29]. Potassium, present at over 1 M concentrations in several hyperthermophiles, has been shown to prevent depurination of DNA at high temperatures [ZO]; however, becausemany hyperthermophiles lack high levels of K+ [ZO], its in vivo role is unclear. Positive supercoiling of DNA may be an important factor stabilizing DNA to high temperatures. A unique Type I DNA topoisomerasecalled reverse DNA gyrase has been found in all hyperthermophilic Archaea and Bacteria [25’,26,27] and has been shown to catalyze the positive supercoiling of closed circular DNA (non-hyperthermophiles contain DNA gyrase, an enzyme that introduces negative supercoils into DNA). For various reasons,in particular its higher linking number, positively supercoiled DNA is more resistant to thermal denaturation than is negatively supercoiled DNA [26,27]. Interestingly, however, at least one hyperthermophilic speciesof Bacteria and possibly one Archaeon contain both gyrase and reverse gyrase [30], leading to some question about the importance of positive supercoiling in DNA thermostability.

to thermophily

For an organismto grow at high temperature its major components, including proteins [2,19’], nucleic acids [20,21] and lipids [ZZ], must be heat stable. The thermostability of enzymes from various hyperthermophiles can be phenomenal, with someremaining active up to 140°C [23]. Although no firm rules governing thermostability are known, structural studies of several thermostable proteins point to a rather smallnumber of noncovalent features, including highly apolar cores, reduced surface-to-volume ratios, decreased glycine contents, and a high number of ionic interactions, that are highly correlated with thermostability [19’,23]. A hydrophobic core undoubtedly helps exclude solvent from the internal regions of the protein, making it internally ‘sticky’ and thus more resistant to unfolding. A small surface-to-volume ratio is ultimately a function of folding and probably improves stability by conferring a compact form on the protein. A reduction in the glycine content of thermostable proteins reduces options for flexibility and thus introduces rigidity, and this, along with the extensive ionic interactions that form a network over the surface of the molecule, helps the compacted protein resist unfolding at high temperature [19’]. In addition to these intrinsic stability factors, protein thermostability can alsobe facilitated by extrinsic factors, such as molecularchaperonins.One very interesting chaperonin,the thermosome[24], has been characterized from Methanopyw

Several hyperthermophilic methanogens of the kingdom Euryarchaeota [7’] contain histone-like proteins; these proteins closely resemble in both structure and function the core histones of Eukarya [28”,29]. Archaeal histones wind and compact DNA into nucleosome-like structures [31’] that maintain DNA in a double-stranded form at high temperatures [32]. Although present in many methanogens, histonesare absent from crenarchaeotal speciesof Archaea [‘29] that include the most thermophilic of all known life forms [6”,7’,10’]. Small DNA-binding proteins like Sac7d from the crenarchaeote Sulfo~obus acidocaldatius, however, are present and may be functional equivalents of histones; Sac7dhasbeen shownto bend and significantly increasethe melting temperature of DNA [33]. The compaction/protection of DNA by histones or Sac7d-like proteins might also explain why similar transcription requirements, including those for a structurally complex RNA polymerase,initiation factors, and a TATA binding protein, occur in both Archaea and Eukarya [25’,28”,29]; presumably these would be needed to gain accessto DNA for transcription and for interactions of the DNA with various regulatory elements.

Halophilic

microorganisms

and their

habitats

Halophilic microorganismsabound in hypersaline lakessuch as the Dead Sea, the Great Salt Lake (Utah), and saltern evaporation ponds. Such lakesare often colored red by dense communities of pigmented microorganisms (halophilic Archaea and/or the B-carotene-rich green alga Dunaliella)

Thermophilic

[X4-36]. Other habitats for hatophitic and halotolerant microorganisms include salted foods and saline soils. Iiatophites are found in at! three domains of life: Bacteria, Archaca, and Eukarya. Some (e.g. the Ractcrium ffuhmu~urs e/wz,iztcr) can adapt to life over a wide salt range, whereas others, such as Archaea of the order FIalobacteriales (‘halobacteria’), are adapted to !ifc in near-saturated brines, and are unable to grow and even die below 1.5-X% NaCt (‘Fable 1). Our understanding of the diversity of hatophitic microorganisms has increased tremendously in past years. On the basis of comparative sequencing of 1hS rKNA the Hatobacteriales appear to be a diverse taxonomic rearrangements have group, and extensive recently been proposed [X7]. ‘l‘he modcrate!?; halophilic Ractcria also rcccntly received the exposure they deserve in a comprehensive review [X3]. Underground salt deposits form interesting environments for halophilic bacteria, and several have been cxptored rcccntty with the idea that they may harbor ‘ancient’ microorganisms that may have survived in a dormant state for hundreds of millions of years. In this regard. a variety of hatophitic Archaea were isolated from the Permian Satado salt formation near Cartsbad, Kcw hlexico, including several previously undiscovered types [39]. Ilowevcr, before it can be concluded that swh isolates arc indeed derived from ancient hypersatine seas, it should he ascertained that the sites have remained undisturbed during geological history and that appropriate aseptic sampling techniques have been used [40]. In fact, cvidence arguing against the antiquity of these organisms has emerged from the fact that l(,S rKNA ,qencs in H~~hmzh isolates from ancient salt deposits are wry similar to those of ‘modern’ isolates [al’].

Molecular

adaptations

to halophilism

‘live fundamenwlty different strategies cnahle microorganisms to cope with osmotic stress. ‘I’hc first. used h); the halobacteria (Archaea) and by a group of anacrobic hatophilic Bacteria. involves accumulation of inorganic salts in the cytoplasm. K+ rather than Na+ is the dominant intracctlular cation. and C- the dominant anion. In the halobacteria the concentration gradient of Na+ over the cytoplasmic membrane is created by action of the Na+/H+ antiporter transporter system. ‘I‘hc energy required to pump Na+ out of the ccl! is supplied by the proton gradient formed during respiratory electron transport or (in some species) by the light-driven proton pump bacteriorhodopsin. I<+ ions probably enter the cell passively in response to the membrane potential. ‘Two inward chloride transporters have been identified in Hcc/ohteri;u~~ the first, halorhodopsin, is tight-dependent, and the second is probably energized by symport with Na+ [3,38,4?.43.44]. ‘l’he presence of molar concentrations of inorganic ions in the cytoplasm requires far-reaching adaptations of the intraccllutar enzymatic machinery [42*,4X44]. Pclost proteins of the halobacteria contain a targc excess of acidic amino acids

and

halophilic

extremophiles

Madigan and Oren

267

and a low number of basic amino acids. ‘I-he high cation concentrations within the cell may in part be needed to shield the negative charges on the protcin surface. In addition, a high content of glutamate may be favorable as glutamate has the greatest water binding capacity of all amino acids, thus enabling the maintenance of a hydration shell. The requircment for high salt concentrations for structural stability can also be attributed to the low content of hydrophobic residues: high salt is needed to maintain the weak hydrophobic interactions [44]. Accordingly most proteins of the halobactcria denature when suspended in solutions containing less than 1-2 \I salt [33]. A thermodynamic ‘salvation-stabilization hypothesis’ has been formulated to explain the stabilization of halophilic proteins in terms of protein-sotvcnt interactions [AS]. According to this mode!, stability of the folded structure involves a network of hydrated ions associated with the acidic residues at the surface of the protein. Site-directed mutngenesis is presently being employed to test the mode! in sctected proteins [4.5]. ‘I‘he spcciat propcrtics of halophitic proteins are nicety itlustrated in a recent study on the glutamate dehydrogenase of H~~/ohtu-int~~st~li~tw~~ [+I”]. A mode! of the enzyme, based on its primary structure and comparison with other glutamate dehydrogenascs, shows the surface of the halophilic protcin to he coated with acidic residues. ‘I‘he surface also displays a significant reduction in hydrophobic character. It was suggested that this was not primarily due to loss of surf&e-exposed hydrophobic residllcs as has previously been lproposcd, but to a reduction in surface-exposed Iysine residues. Another enzyme rccent!y characterized is dihydrofolatc reductase of H~~/of~mxuoltmii [47”], whose structure was elucidated at 2.6 ‘4 resolution by X-ray crystallography ‘I’his enzyme requires only moderate salt concentrations, and retains its activity at nionovalcnt cation concentrations as low as 0.5 1’1. Here also clusters of negative charges were identified at the surface of the protein. A theoretical analysis of the contribution of electrostatic interactions in H~Iomruh rmrnLvnol;tz~j ferredoxin and malate dehydrogenase showed that repulsive interactions between acidic residues at the protcin surface were 3 major factor in destabilizing these hatophilic proteins in low salt conditions. It was suggested that the acidic residues might bc important in preventing aggregation rather than contributing to intrinsic protein stability [3X”]. ‘l’hc second option for adaptation to life at high salt, rcalizcd in most hatophilic Bacteria. cukaryotic algae and and in the halophilic methanogenic Archaea, fungi, involves the maintenance of a cytoplasm tow in salt but high in other solutes. Organic osmotic ‘compatible’ solutes provide osmotic balance while enabling activity of ‘conventional’. non-salt-adapted enzymes. Examples of such solutes are glycerol (in Lhdie/h and certain fungi), glycine bctaine (in halophilic photosynthetic Hactcria), and ectoine (synthesized by many autotrophic and hcterotrophic Bacteria) [49]. Compatible solutes are polar, highly lvater-soluble molecules that are uncharged or zwit-

268

Ecology

and

industrial

microbiology

terionic at physiological pH. Their intracellular concentrations are regulated according to the external salt concentration. The use of organic osmotic solutes thus bestows a high degree of flexibility and adaptability to the cells. Compatible solutes are strong water structure formers and, as such, are probably excluded from the hydration shell of proteins. They stabilize proteins by preventing unfolding caused by heating, freezing, or drying [49]. The genes involved in the biosynthesis of ectoine by ffalomonas elongata and Marinococcus tialophihs have recently been cloned and characterized [50”,.51”], and osmoregulated expression of the M. Aalophihs genes in E. co/i wasdemonstrated [Sl”]. H. eloongata can be exploited to produce large quantities of ectoine and hydroxyectoine (useful as moisturizers in skin care products) in a process called ‘bacterial milking’, in which dense cultures are exposed to an osmotic down-shock, causing excretion of excess osmotic solutes. Addition of salt to the culture induces resynthesisof ectoine and hydroxyectoine, whereafter the processcan be repeated [SZ’].

4.

Stetter 1996,

prokatyotes.

5.

Brock TD: Tbermophilic Microorganisms Temperatures. New York: Springer; 1978.

FEMS and

Microbial

Rev

Life at High

Stetter KO: Volcanoes, hydrothermal venting;and the origin of life. In Volcanoes and the Environment. Edited by Marti J, Ernst GJ. Cambridge: Cambridge University Press; 1999:in press. An excellent and very comprehensive review on the microbial diversity and characteristics of the habitats of hyperthermophiles. Contains an excellent collection of photos of thermal habitats, both terrestrial and submarine, and discussion of what hyperthermophiles have taught us about the origin of life. l

6. *

7. .

Stetter KO: Hyperthermophiles: isolation, classification, and properties. In Exfremophiles - Microbial Life in Exfreme Environments. Edited by Horikoshi K, Grant WD. New York: Wiley; 1998:1-24. An excellent review of the diversity, phylogeny, and physiology of hyperthermophiles 8.

Barns SM, Fundyga archaeal diversity spring environment.

RE, Jeffries MW, Pace NR: Remarkable detected in a Yellowstone National Park hot froc Nafl Acad Sci USA 1994, 91 :I 609-l 613.

9. ..

Hugenholtz P, Pitulle C, Hershberger KL, Pace NR: Novel division level bacterial diversity in a Yellowstone hot spring. J Bacferiol 1998, 180:366-373. Another example of molecular microbial ecology, this time with a focus on the Bacteria rather than the Archaea. This paper updates the status of the phylogenetic tree of Bacteria, indicating that at least 35 major phylogenetic groups of Bacteria exist, and shows that in thermal environments great microbial diversity is present in the domain Bacteria as well as Archaea. 10. .

Conclusions In recent years several new speciesof hyperthermophilic and halophilic prokaryotes have been isolated and considerable progress made in the study of molecular mechanismsthat enable their proteins to function under extreme conditions. A major unanswered question in the microbiology of hot environments is “What is the upper temperature limit for life?” Several factors suggestthat this will be 140-15O”C, and cultures of such ‘super hyperthermophiles’ would indeed be very exciting. Researchon the halophiles is just beginning to reveal the importance of intrinsic structural factors in protein stability and much more data are also needed on the mechanisms by which osmotic solutes stabilize enzyme activity. Our understanding of the ecology of hyperthermophilic and halophilic prokaryotes lags far behind our knowledge of their physiology and molecular biology, and it is hoped that future work will focus on the community structure and ecological interactions of these fascinating organisms in their extremely hot or salty habitats.

Blijchl E, Rachel R, Burggraf S, Hafenbradl D, Jannasch HW, Stetter KO: Pyrolobus fumarii, gen. and sp. nov. represents a novel group of Archaea, extending the upper temperature for life to IlF’C. Exfremophiles 1997, 1 :14-21. A description of the most thermophilic of all prokaryotes known to date. Organisms like fyrolobus may revise microbiological dogma on what constitutes a correct autoclave temperature! 11.

Kurr M, Huber R, KBnig H, Jannasch HW, Fricke H, Trincone A, Kristjansson JK, Stetter KO: Methanopyrus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 11O’C. Arch Microbial 1991, 156:239-247.

12.

Harmsen HJM, Prieur D, Jeanthon C: Distribution of microorganisms in deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture thermophilic subpopulations. Appl Environ Microbial 1997, 6312076-2883.

13.

on extremophilic prokaryotes in the laboratory of MT by National Science Foundation Gram OPP 9809195. by grant no. 95-00027 from the United States-Israel Foundation (BSF, Jerusalem).

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