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Volume 87



Volume 87, Number 1 Winter 2000

Annals of the

Missouri Botanical Garden

The Annals, published quarterly, contains papers, primarily in systematic botany contributed from the Missouri Botanical Garden, St. Louis. Papers originating out side the Garden will also be accepted. All manuscripts are reviewed by qualified independent reviewers. Authors should write the Managing Editor for informatior concerning arrangements for publishing in the Annals. Instructions to Authors an printed in the back of the last issue of each volume and are also available online a www.mobot.org (through MBG Press).

Editorial Committee

Victoria C. Hollowell


Missouri Botanical Garden

Amy Scheuler McPherson

Managing Editor, Missouri Botanical Garden

Diana Gunter Associate Editor, Missouri Botanical Garden

Vicki Couture Senior Secretary

Barbara Mack

Administrative Assistant

Ihsan A. Al-Shehbaz Missouri Botanical Garden

Gerrit Davidse

Missouri Botanical Garden

Roy E. Gereau

Missouri Botanical Garden

Peter Goldblatt

Missouri Botanical Garden

Gordon McPherson Missouri Botanical Garden

P. Mick Richardson Missouri Botanical Garden


van aer



Missouri Botanical Garden

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The mission of the Missouri Botanical Garden is to discover and share knowledge about plants and their environment, in order to preserve and enrich life.

This paper meets the requirennents of ANSI/NISO Z39.48-1992 (Permanence of Paper).

Volume 87 Number



of the


Botanical Garden



P. Mick Richardson'^

Recent years have seen many new discoveries in and one wonders if there are more of them around the plant, animal, and other kingdoms. Can we es- the world to be discovered. The finding of this sec- timate how many more organisms are out there to ond species of coelocanth was headline news to bi- te discovered? An international group of experts has ologists, as was the discovery of the Wollemi Pine been invited to St. Louis to give their thoughts and and several new large mammals, discussed in the predictions about this intriguing area of biology. The following pages. However, the regular and contin- symposium will consist of an exciting set of presen- ued reporting of new species usually attracts less tations, ranging from flowering plants in the U.S., attention. For example, one of my former students Australia^ and the tropics, to freshtvater fishes, mam- recently published five new species in the brome- mals, and last, but not least in every sense, extre- Had genus Cryptanthus (Ramirez, 1998). To my mophiles and other bacteria. Flyer advertising the mind, such a publication may actually be more im-

45''' Annual Systematics Symposium.

portant than the discoveries which make headline

In February 1998 I was in London, and purely news because the new species of Cryptanthus are by chance I was able to attend a talk at the Linnean the result of full and detailed revisions of the genus Society on coelocanths, expertly delivered by Pete based on field, herbarium, and laboratory studies.

Ives in the follow-

Forey. Later that year my wife and I were impressed The readers can judge for th

by a coelocanth specimen in a delightful marine ing papers that comprise the published proceedings

science museum in Tulear, a specimen caught lo- of the symposium.

cally off the southwest coast of Madagascar and

The 1998 Annual Systematics Symposium dif- thought to have been swept southwards from the fered from the usual format in that there were ten populations around the Comoro Islands. However, speakers rather than the usual seven. This allowed coelocanths have recently been found in Indonesia, for a greater diversity in composition of the program

' This and the eight articles that follow it are the proceedings of the 45th Annual Systematics Symposium of the Missouri Botanical Garden, Our Unknown Planet: Recent Discoveries and the Future. The symposium was held 9-10 October, 1998, at the Missouri Botanical Garden in St. Louis, Missouri, U.S.A.

The symposium was supported in part by the National Science Foundation under grant number DKB-942()14(). I thank Peter Raven for helping to select a fine diversity of speakers, Kathy Hurlbert and her expert staff for wonderful help in organizing and administrating the symposium, Barbara Alongi for her fine illustration used for the cover of the symposium brochure, and the symposium speakers for being such a pleasant group of scientists.

^Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, U.S.A.

Ann. Missoi ri Bot. Gard. 87: 1-2. 2000.


Annals of the

Missouri Botanical Garden

and allowed a wider variety of organisms to be cov- tegrate their analyses and discuss eritena for es- ered in some detail. The morning session began tablishment of higher taxa.

with Michael Madigans talk on prokaryotic^ organ-

The plant talks t^ame after lunch. Iain Prance

isms, a group that constitutes the bulk of evolu- infonned the audience (and now the readers) that tionary diversity on Earth and which is of increas- the number of angiospernis is currently undcit^sti- ing interest for use in biotechnology and related mated, and he confidently predicliMl thai then^ are areas. Think about where systematics would be in fa<;t l>etween 300,000 and 320,000. lie used without the PCR methodology so necessary for cur- specific examples of the discovery of palms in Mad- rent molecular systematics techniques. Literally, agascar and other areas, as well as detailed studies

the book on molecular systematics of plants had to «f ^H genera in areas in Brazil and Brunei, to de- be rewritten within six years (Soltis et al., 1992, velop his case for an intensification of [\\e rate of 1998). Microbes were followed by Richard Brusca's collection to confirm his predictions before it is too fascinating talk on arthropod diversification, and ^^^^- Barbara Briggss talk on l)()tanical discoveries this made me think of eating deliciously tasty large >" Australia contrasted th(^ media attention given to crabs caught in the River Jurua in Acre, Brazil. ^^^ <hscovery of a new genus of conifers compared Next was Ebbe Nielsen^s discourse on insects, un- ^^ ^^''' uncharismatic discovery of 61 new species fortunately not included in the published proceed- ^" ^^^ Restionaceae and allied families. Finally, ings. Our current knowledge of freshwater fishes ^'""'^'''''^ ^^*^^' "'^*'*' ^^^ suq,nsing annoimcement was the subject of John Lundberg and his col- that the rate of discover) of new plants in ^

Kc *! A r i_ ed States and Canada has been constant for the past

boutn Amencan iishes are so mcom- , . , . __



pletely known, I wonder if the diversity of fishes we ate alongside the aforementioned crabs may

have been species new to sci

. Fortunately, they

century and shows no evidence of tapering off.

Mike Donoghue gave a vt^ry enteilaining and stimulating after-dinner talk, emphasizing that the current age of discovery may be different from ear-

were photographed before they went into the fryins; i- i » •. i a i i n .

I^ . . ^i*^r ones, but it is uoln riciier and more illuminal-

pan, leaving some clues at least to their existence. t* .u i * r ii * .* * * * *i

. , . . if^g- 't IS the duty oi ail systematists to captun^ tn<

Last in the animal line of talks was John Mac-

imagination of other scientists and, even more im-

Kinnon, who told us about new ungulates being dis- po^-tantly, the public^ at large, covered in Vietnam and his predictions about where future finds will likely be made. The morning

Lileralure (]ited

session ended with some wonderful video footage Marf^niHs, L. 1998. Syiuhioiit Plaiui: A New Look al Evo-

1 i-j r T n* 1- . 11-111 hilion. Masic Books, New York.

and slides trom Lynn Margulis, not published here, & l- m c i . ww^o i l i a h

■^ ^ ' ^ ' <\ K. V. Schwartz. I99H. hvc Kmg<loms: An II-

but see her books Five Kingdoms: An Illustrated lustraicd Guide to tin- Ph>la of Life <mi Earth. Freeman,

Guide to the Phyla of Life on Earth (Margulis & San Fraiuiseo.

Schwartz, 1998) and Symbiotic Planet: A New Look R'^n»i'*^^" '• M- l'^^^^- ''ivc new species oi CrypianihiLs

^ , . .,- ,. ir\r\rt\ Tvn -i (Hronieliaeeae) ^ind some nomen<*lalural novelties. Har-

at tvolutwn (Margulis, iWo). While not everyone ^.^^.^j p. p^^^ ■^. 2n-224

will agree with Margulis's concept of monophyly, Soliis, I'. S., D. K. Soltis & J. J. Doy]<^ (Editors). 1992.

there is no denying that she has added a very in- Moleeular Systematics of Planls. Cliajunan *S Hall, New

teresting viewpoint to overall discussions of biodi- o i "^ "i^ i^ n c c i i> i i i^ i /i- i- n ..uu,

. , , . , , Soltis, D. E., P. S. Soltis cK J. J. Doyle (Editors). 1998.

versity, and at the same time she makes an urgent Molecular Syst<'niaties of rianls ll': DNA Stqianein^.

appeal for all biologists and paleontologists to in- Kluwer, lioston


Michael T. Madigan



Many prokaryolic microorganisms inhabit "extreme environments" habitats in which some chemical or physical variable(s) differs significantly from that found in habitats that support plant and animal life. Great strides have been made in recent years in the isolation and characterization of extremophilic prokaryoles, and many of them turn out to have fascinating metabolic properties and interesting evolutionary histories. Prokaryotes that grow at very high tem- peratures are perhaps the most dramatic in these regards, as all cellular components need to be made heat stable and their evolutionary position is that of the least evolved of all known life forms. As our knowledge of bacterial diversity improves, primarily from the introduction of molecular tools for assessing bacterial phylogeny and diversity and from new advances in isolation and laboratory culture, it is becoming clear that the bulk of evolutionary diversity on Earth does not reside in plants and animals, but instead in the invisible prokaryotic world. There is now great interest in mining the diverse genetic resources of Earth's smallest cells for use in biotechnology and related areas.

Key words: extremophilic bacteria, evolutionary history, microbial diversity, prokaryotes.



Since the days almost 100 years ago when Robert bacteria. This giant 1

Koch and his associates isolated the first pure cul- tools of molecular bic

tures of bacteria, microbiologists worldwide have development of rapid gene sequencing methods and

been isolating laboratory cultures of literally thou- powerful algorithms for the comparative analysis of

sands of different bacteria. These include, of course, nucleic acid sequences. But for these advances to

most of the causative agents of infectious diseases, impact microbial evolution, a gene or genes that but more important from the standpoint of the web reflected the evolutionary history of an organism of life on Earth, many of the bacteria that carry out had to be identified. Such an evolutionary "Rosetta critical chemical reactions that form the "life sup- Stone" had long been sought, but not until the ad- port" system for plants and animals (Madigan et al., vent of comparative ribosomal RNA sequencing as 2000). Despite the diversity of organisms that are a rapid and specific means for deducing bacterial already known, it is now clear that microbiologists phylogenies (Woese, 1987) did microbiologists have have only seen the tip of iceberg; most microorgan- tl isms that exist in nature, in particular the bacteria, ural fashion the way botanists and zoologists had have not yet been obtained in laboratory culture! classified their subjects for over a century using Indeed, with the help of new molecular tools micro- primarily phenotypic characteristics such as bones biologists have explored a variety of microbial hab- or leaf arrangements as evolutionary guideposts.


itats and have detected not only new species of bac-

Two key concepts have emerged from compara-

teria, but new genera, families, orders, and even tive molecular sequencing of ribosomal RNAs: (1) phyla (Bams et al., 1994; Hugenholtz, el al., 1998). that cells evolved along three major lineages, the Imagine finding a new phylum of plants or animals Bacteria, the Archaea, and the Eukarya, instead of

today! The challenge for microbiologists now is to

prokaryotes and

isolate these organisms, learn alx)ut their basic bi- 1); and (2) that the evolutionary difference between ology, and harness their vast genetic resources for a mouse and an elephant (or between Chlorella and the benefit of mankind. Trillium, for the more botanically oriented) pales by

comparison to the evolutionary distance between virtually any two common soil bacteria you might want to mention, like Pseudomx>nas and Bacillus. The first of these conclusions, that prokaryotic crobial diversity in recent years because of the life contains two major evolutionary lineages, is new-found ability of microbiologists to experimen- slowly but surely becoming mainstream thinking

among microbiologists, and is even gaining support

A Natural Picture of the Bacterial World

Great excitement has pervaded the field of mi-


' The research of M. T. Madigan is supported by National Science Foundation grant OPP 980919S. ^ Department of Microbiology and Center for Systematic Biology, Mailcode 6508, Southern Illinois University, Car- bondale, Illinois 62901-6508, U.S.A. madigan@micro.siu.edu

Ann. Missouri Bot. Card. 87: 3-12. 2000


Annals of the

Missouri Botanical Garden


















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E o







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Volume 87, Number 1 2000

Madigan Extremophilic Bacteria


from macrobiologists as evidenced by the inclusion relationships of prokaryotes (Fig. 1), but can also

of this concept in recent biology textbooks (Raven use phylogenetic information to construct highly

& Johnson, 1999; Raven et al., 1999). However, specific nucleic acid probes as a means of identi-

the second conclusion, that morphologically quite fying and tracking specific microorganisms in the

different plants or animals can be extremely closely environment. A natural application of this technol-

related in a molecular evolutionary sense, has been ogy has been to take these tools into various ex-

for many a harder pill to swallow. If one steps back treme environments and probe for the diversity of

for a moment and considers that it is not the evo- microbial life therein. The fallout from these stud-

lution of the mouse and the elephant, or the alga ies, which historically followed by many years more

and the flowering plant, as intact entities, that mo- classical enrichment and isolation approaches, has

lecular sequencing speaks to, but instead, the evo- been an awareness that extreme environments are

lutionary history of the cells that make them up, it "ot a place for "hangers on," but instead are hab-

is easier to understand why the bulk of evolutionar>' itats that flourish with microbial life, especially pro-

hange has occurred in the prokaryotic worid; pro- karyotes (Bams et al., 1994; Horikoshi & Grant,

1998; Hugenholtz et al., 1998). The rest of this

while the mouse and the elephant have only very P^P^^ ^^1^ try to introduce the reader to some of

kar}otes have existed for some 3.8 billion years,

recently evolved and diverged.

these organisms and their homes, and discuss what

Prokaryotes ruled the Earth for at least 2 billion laboratoiy studies of these remarkable prokaryotes years before the modem (organelle-containing) eu- ^ave revealed for our understandmg of the physi-

karyotic cell appears in the fossil record. And metazoans (multi-celled plants and animals) have

only existed for some half billion years or so. So by EXTF^EMK ENVIRONMENTS AND EXTREMOPHFLKS

ochemical limits to Hfe.

the time the stage was set for what botanists and zoologists consider the "evolutionary diversification of plants and animals," most of cellular evolution had already occurred. Diversification of the mouse and the elephant, for example, was simply a matter

Microbiological examination of extreme environ- ments has revealed many new prokaryotes. By "ex- treme environment" here, it is meant an environ- ment that humans would consider extreme or uninhabitable: extremes of heat or cold, pH, salin- ity, pressure, and even radiation. As previously mentioned, extreme environments are inhabited by

diverse populations of microorganisms, most of

and the elephant are virtually identical organisms. iii ii*t i*u f

^ y D which have evolved to live only in the presence oi

In contrast to higher organisms, prokaryotes have ^j^^ ^^^reme. These organisms are the

of arranging cells in different ways to yield what appears to the eye to be highly divergent organisms. But in terms of their evolutionary' history, the mouse


had more evolutionary time to show great genetic ^j^j^^^,, (Horikoshi & Grant, 1998; Madigan &

divergence. However, unlike metazoans, evolution- j^^^^^ ^997. Madigan et al, 2000; Madigan &

ary change in prokaryotes is not manifest in mor- q^.^^^ ^g^y Several classes of extremophiles are

phological variation. For whatever reason(s), bac- recognized in microbiology, and laboratory cultures

teria maintained a ver)^ small size and changed ^f representatives of each class are known (Table

relatively little (compared with metazoans) in mor-

1). Organisms in each class are denoted by a de-

phology through billions of years of evolutionary scriptive term, usually a word with Greek or Latin history. But that is not to say they did not evolve. ^^^^s followed by the combining f

orm "phile,"

Molecular sequencing tells us that they have in- Greek for "loving." Thus there are thermophiles and

deed evolved but that the product of this evolution- hyperthermophiles (organisms growing at high or

ary change is invisible— instead of big changes in y^^y high temperatures, respectively), /;.sjc/iro/>/zi7e.s

size or shape, evolutionary change in the prokary- (organisms that grow best at low temperatures),

otes focused on metabolic diversity and the genetic acidophiles and alkaliphiles (organisms optimally

capacities to explore and eventually colonize every adapted to acidic or basic pH values, respectively),

conceivable environment on Earth, including ex- barophiles (organisms that grow best under pres-

treme environments. Thus we must go to the genes sure), and halophiles (organisms that require NaCl

of the prokaryotes to see their true phylogenetic for growth) (Table 1). Instead of trying to be inclu-

diversification, and with advances in nucleic acid sive here, as literally hundreds of different species

sequencing, this world is now beginning to open up could be included, the organisms listed in Table 1

(Madigan et al., 2000). are the current "record holders" in each of the

Using comparative ribosomal RNA sequencing tremophile categories. The column of most interest

microbiologists can now not only construct natural in Table 1 is the one labeled "optimum," for here


Annals of the

Missouri Botanical Garden

Table 1. Classes and examples of extremophiles\










Hyperthemiophile Pyrulobus fumarii




Pularumonas vacuolata

90°C OX








Picrophilas oshimae Natronobacterium grego- ryi

0.06 8.5

0.7 (60T)* 10 (20% NaCl>'


Salt (NaCl)

Barophile Halophile

MT41 (Mariana Trench)'' 500 atm

Halobacterium saUnarum


700 atm (4X) 25%

113X 12°C

4 12

1000 atm

32% (saturation)

^ In each category the organism listed is the current "record holder" for requiring a particular exl growth. '• Strain MT41 does not yet have a formal genus and species name and is also a psychrophile. * P. oshimae is also a thermophile, growing optimally at 60X. *' N. gregoryi is also an extreme halophile, growing optimally at 20% NaCI.

condition for

it becomes cleeir that these organisms are not mere- isms set the stage for the evolution of modem life ly tolerating their lot, but that they actually do best forms, in their punishing habitats; indeed most actually

require their extreme condition(s) in order to repro- \jyy. at High Temperature

duce at all.

Extremophiles are of interest to both basic and

Although thermophilic bacteria (organisms with

applied biology. In a basic sense, these organisms growth temperature optima between 45^C and 80°C)

hold many interesting biological secrets, such as have been known for over 80 years, hyperthermo-

the biochemical limits to macromolecular stability philic bacteria organisms with optima above

and the genetic instructions for constructing mac- 80°C have only been recognized more recently

romolecules stable to one or another extreme (Ma- (Stetter, 1996). Following the pioneering work of

digan & Oren, 1999). But in an applied sense, Thomas Broc;k in the 1960s and 1970s (reviewed

these organisms have yielded an amazing array of in Brock, 1978), Karl Stetter and co-workers at Re- enzymes capable of catalyzing specific biochemical gensburg (Germany) have proceeded to isolate over reactions under extreme conditions (Adams & Kel- 30 genera (> 70 species) of hyperthermophiles.

ly, 1995). Such enzymes have served as grist for Brock was the first to demonstrate, often using sim-

industry in applications as diverse as laundry de- pie but ingenious field experiments, that bacteria

tergent additives (proteases, lipases) and the ge- were present in boiling hot springs in Yellowstone

netic identification of criminals {Taq DNA poly- National Park (Brock, 1978). By contrast, Stetters

merase and its use in the polymerase chain group, whose focus has been on isolation and cul-

reaction, PCR). ture, has isolated many of the hyperthermophiles

Another important realization that has emerged known today, including Pyrolohus fumarii^ a re-

from the study of extremophiles is that some of markable prokaryote that can grow up to 113X (Ta-

these organisms form the cradle of life itself. Many ble 1, Fig. 2) (Blochl et al., 1997).

Thermophilic microorganisms can be isolated lie close to the "universal ancestor" of all extant from virtually any environment that receives inter- life on Earth (Fig. 1). Thus, an understanding of mittent heat, such as soil, compost, and the like.

extremophiles, in particular the hyperthermophiles.

the basic biology of these organisms is an oppor- But hyperthermophiles thrive only in very hot and

tunity for biologists to "look backward in time" so constantly hot environments, including hot springs,

to speak, to a period of early life on Earth. This both terrestrial and undersea (hydrothermal vents),

exciting realization has fueled much research on and active sea mounts, where volcanic lava is emit-

these organisms in order to understand the nature ted directly onto the sea floor (Stetter, 1999). It is

of primitive life forms, how the first cells "made a also strongly suspected, and some supportive evi-

living" in Earth's early days, and how early organ- dence exists, that hyperthermophiles reside deep

Volume 87, Number 1 2000

Madigan Extremophilic Bacteria



T * f

Figure 2. Transmission (^Icclron mirrogra[)h of a cell i}{ Pyrolohus fHiruird, llic riiosi tlieniio[)liili(; of all known living organisms. PyrolobiLs fnmani grows opiirtially at \{Y}\] and < an grow at up to I 1.5'^C. Kv<^n higher leniper- alures are lolcratetl hut <lo not support growth. Mi<Tograph courtesy of HeinhanI Karhf^I, liniversilat R(*genshurg.

the chimneys, which are often only about 0.5 thick, show a temperature gradient from about 300*^C inside to 2°C outside. Because prokaryotes are so small, microenvironments differing in tem- perature exist across the chimney wall leading to ideal habitats for various species of heat-loving bacteria.

Using nucleic acid probe technology several

morphological types of bacteria have been detected in hydrothermal vent chimney walls (Harmsen et al., 1997), suggesting that these compact thermal gradients may contain many different microbial populations in addition to those already isolated. And for my botanical friends reading this paper, I would be remiss if I did not point out that P.famarii and M, kandleri are good examples of primary pro- ducers totally divorced from sunlight, a capacity

widespread in the microbial world. Besides growing at almost unbelievably high temperatures, R fu- marii and M. kandleri are also autotrophs, capable of growing in a simple anaerobic mineral salts me- (Hum supplied with CO2 and H^; neither sunlight nor a key product of photosynthesis, O2, is required within the earth, Hving a buried existence and re- for either organism. Indeed, it has been hypothe- lying on geoihermal heat for their metabolic activ- sized that long before the process of photosynthesis

ities and reproduction (Stetter, 1999).

evolved, anaerobic H^-based chemolithotrophy was

The most extreme of known hyperthermophiles, the major means by which new organic material those with temperature optima above lOO^C, have was synthesized on Earth (Madigan et al., 2000).

conu! from submarine hydrothermal vents (Stetter,

For an organism to grow at high temperatures.

1996, 1999), and examples include P. fumarii especially as high as those of the hyperthermo-

(Bl(k:hl et al., 1997, and Fig. 2) and the methano- philes discussed here, all cellular components, in-

gen Methanopyrus kandleri (Kurr el al., 1991). Both eluding proteins, nucleic acids, and lipids, must be

of these amazing prf)karyotes are members of the heat stable (Adams & Kelly, 1995; Ladenstein &

Archaea (Fig. 1) and are chcmolilhotrophs (organ- Antranikian, 1998; Wiegel & Adams, 1998; van de

isms that use inorganic com[)ounds as energy Vossenberg et al., 1998a). The thermostability of

sources), using molecular hydrogen, H^, as their enzymes from various hyperthermophiles, referred

electron donor (energy source), reducing either to as extremozynieSj has been documented, and

NOi (P. fumarii) or CO^ {M. kandleri) as electron some have been found to remain active up to 140°C

acceptors to grow by anat^robic respiration (Madi- (Adams & Kelly, 1995). The structural features that

gan et al., 2000; Sti^ttcr, 1999). Besides requiring dictate thermal stability in proteins are not well

substantial heat for growth, these bacteria can sur- understood, but a small number of noncovalent fea-

vive temperatures substantially above their upper tures seem characteristic of thermostable proteins.

growth temp<'ratun» limits, making a conventional These include a highly apolar core, which appar-

autoclave regimen (15 min. at 121^) insufficient ently makes the inside of the protein "sticky" and

for sterilizing cultures of either species!

thus more resistant to unfolding, a small surface-

Both P, fumarii and M. kandleri originated from to-volume ratio, which confers a compact form on

hydrothermal vent chimneys (Blochl et al., 1997; the protein, a reduction in glycine content that

StettiT, 1999). These cire precipitated iron mineral tends to remove options for flexibility and thus in-

deposits that form as extremely hot water (up to troduce rigidity to the molecule, and extensive ionic

400°C) containing various minerals emerges from bonding across the protein's surface that helps the

deep-sea h)(lroth(*rmal vents (note that although compacted protein resist unfolding at high temper-

this water is superheated, il does not boil because ature (Ladenstein & Anthranikian, 1998). In ad-

of the hydrostatic pressure of the water column, dition to these intrinsic stability factors, special

usually 2000-3000 m, that overlies these vents). proteins called chaperonins are synthesized by hy-

Althoiigh the water that

rges is too hot for life, perthermophiles. Chaperonins function to bind heat


Annals of the

Missouri Botanical Garden

denatured proteins and refold them into their active the opposing hydrophobic residues from each layer

form. The thermosome is a type of chaperonin that of the

brane together (van de Vossenberg et al.,

is widespread among hyperthermophiles capable of 1998a). This forms a lipid monolayer instead of a

growth above 100°C, like R fumarii and M. han- bilayer, and prevents the membrane from melting

died (Stetter, 1999).

at high temperature. Although the precise chemis-

Several factors may combine to prevent DNA try of lipid monolayer membranes can vary some-

from melting in hyperthermophiles. However, the what from species to species, they are common

two most important features appear to be the en- among hyperthermophiles and are likely an impor-

zyme reverse DNA gyrase, which catalyzes the pas- tant evolutionary response to life at high tempera-

itive supercoiling of closed circular DNA (by con- ture. trast, nonhypcrthermophiles contain DNA gyrase,

an enzyme that supercoils DNA in a negative twist- Lij,^ ^^y l^^ Temperatures ed fashion), and various types of DNA binding pro- teins, Including histone-like proteins (Madigan & How about life at the other end of the thermom- Oren, 1999; Pereira & Reeve, 1998). For various eter? Cold environments on Earth are actually physicochemical reasons, positively supercoiled much more common than hot ones. For e*xample, DNA is more resistant to thermal denaturation than the oceans, which make up over one half the is negatively supercoiled DNA. And the fact that Earth s surface, maintain an average temperature of reverse gyrase seems to be the only protein thus far about 2'^C. And vast land masses are intermittently found universally among hyperthermophiles (re- cold and in some cases permanently cold, or even gardless of their metabolic pattern) (Madigan & frozen. However, cold temperatures are no barrier Oren, 1999) points to an important role for it in the to microbial life, as various microorganisms flourish

heat stability of DNA.

in cold environments, even in ice (Horikoshi &

Several hyperthermophiles contain DNA binding Grant, 1998; Madigan & Marrs, 1997). Many mi-

proteins that appear to play a role in maintaining croorganisms have been isolated capable of growth DNA in a double-stranded form at high tempera- at refrigerator temperatures (4— 8X). These are usu- ture. Some of these proteins are stnacturally related ally psychrotolerant, meaning that although they

to the core histones of eukaryotic cells and function capable of growth in the cold, they grow better at

to wind and compact the DNA into rmcleosome-like warmer temperatures, usually 25— 35°C. True psy-

structures (Pereira & Reeve, 1998). Others have no chrophiles, defined as microorganisms that grow best

structural relationship to histones but when bound at 15°C or lower, are usually only present in per-

to DNA alter its structure in such a way as to sig- manently cold environments like the Arctic, or in

nificantly raise its melting temperature (Madigan & particular, the Antarctic (Horikoshi & Grant, 1998).

Oren, 1999). It is likely that the combination of A variety of microorganisms including algae and

positive supercoiling of DNA along with proteins diatoms have been found in Antarctic sea ice

that prevent DNA melting is a major solution to the ocean water that remains frozen for much of the

maintenance and integrity of DNA in hyperther- year. Sea ice is the habitat for one well-character-


Heat can also affect membrane stability. As all name indicating its affinity for cold temperatures

biologists know, in organisms living at moderate (Irgens et al., 1996). Polaromonas vacuolata grows

temperatures cell membranes are constructed along optimally at 4°C and finds temperatures above 12°C

the typical "lipid bilayer" model: hydrophobic res- too warm for growth (Table 1). Other psychrophiles

ized bacterium, Polaromonas vacuolata^ the genus

idues (fatty acids) inside oppose each other and are known, but because some of them appear to be

retain an affinity for one another while hydrophilic very sensitive to warming, great care must be taken

residues (such as glycerol phosphate) lie at the sur- in their isolation and culture to prevent killing them

face of the environment and the cytoplasm, respec- off at temperatures as low as room temperature, tively, maintaining contact with the aqueous phase. An understanding of the biochemistry and mo-

If one applies sufficient heat to such a membrane lecular biology of psychrophilic bacteria is in a

hitecture the two leaflets of the membrane will much earlier stage than that of the hyperthermo-

pull apart, leading to membrane damage and cy- philes. From what is known about the biochemistry

toplasmic leakage. To prevent this from occurring of psychrophiles, it appears that their proteins func-

at very high temperatures, hyperthennophiles have tion optimally at low temperatures because they are evolved a novel membrane structure. Instead of constructed in such a way so as to maximize flex-


rmmg a

brane as a lipid bilayer, as just dis- ibility; this is essentially the opposite strategy- from cussed, some hyperthennophiles chemically bond that of hyperthermophiles (see earlier). Moreover,

Volume 87, Number 1 2000

Madigan Extremophilic Bacteria


proteins frf)m psychrophiles are typically more po- (< pH 1) solfatara in Italy, and the organism has lar and less hydrophobic than proteins from hy- clearly evolved to require these highly acidic con- perthermophiles, a fact that undoubtedly also as- ditions for its very existence.

sists in their relative flexibility.

Interestingly, however, acid-loving extremo-

Besides keeping their enzymes functional, psy- philes, even those as extreme as P. oshimae, cannot

chrophiles have other biological problems to con- tolerate great acidity inside their cells, where it

tend with, transport of nutrients across the mem- would destroy such important molecules as DNA.

brane being chief among them. However, just as They thus survive by keeping the acid out. The

margarine, with its higher content of unsaturated internal pH of P, oshimae is about pH 5, and it is

fats, can stay softer than butter at cold tempera- the cytoplasmic membrane of this organism that

tures, psychrophiles regulate the chemical compo- keeps protons from passively entering the cell.

sition of their membranes, including in particular However, studies of the R oshimae membrane have

the length and degree of unsaturation of fatty acids, shown that it can only retain its integrity in acidic

to keep them sufficiently fluid to allow for transport solutions; above an external pH of about 4 the R

processes, even at temperatures below freezing oshimae membrane spontaneously disintegrates.

(Horikoshi & Grant, 1998). Applications of en- Major unanswered questions concerning the metab-

zymes from psychrophiles include the cold food in- olism of R oshimae and other extreme acidophiles

dustry, where enzymes that work at refrigerator tem- concern how they generate a proton motive force

peratures are sometimes desirable, as well as during respiration and related issues of bioenerget-

producers of cold-water laundry detergents (see ics involving membrane-mediated proton translo-

more on this below).

cation (van de Vossenberg et al., 1998b).

Various acid-tolerant enzymes from acidophiles,

Life in Bath hy Acid or S()D\ primarily ones located on the cell surface or ones

excreted from the cell into the acidic milieu, have Many extremophiles have evolved to grow best been studied and potential industrial applications at extremes of pH: these are the acidophiles and identifie<l. These are primarily as animal-feed sup- the alkaliphiles (Horikoshi & Grant, 1998). Al- plements where the enzymes function to break though extremely acidic or alkaline (below pH 3 or down inexpensive grains to more nutritionally ben- above pH 10) habitats are rare on earth, in such eficial forms directly in the animal's stomach. Such environments one can find a variety of microorgan- enzymes have been widely used in the poultry in- isms thriving in chemistry the equivalent of battery dustry and have been shown to reduce feed costs acid or soda-lime. Highly acidic environments can and the time necessary to get birds to market.

result naturally from geochemical activities, such

Extreme alkaliphiles live in soils laden with soda

nearer to

as from the oxidation of SO2 and H^S produced in (natron) or in soda lakes where the pH can rise to hydrothermal vents and hot springs, and from the as high as 12. N atronobacterium gregoryi (Table 1), metabolic activities of certain acidophiles them- for example, was isolated from Lake Magadi, a soda selves. For example, the iron sulfide-oxidizing bac- lake located in the Rift Valley of Africa; A^. gregoryi terium Thiohacillns ferrooxidans can generate acid grows optimally at a pH of about 10 (Table 1) (Hor- by oxidizing Fe-^ to Fe^^, the latter of which pre- ikoshi & Grant, 1998). In the opposite scenario cipitates out as Fe(OH)j (Fe^"^ + 3H;^0 > Fe(OH)^ from the acidophiles, alkaliphiles have to contend + 3H'), or by oxidizing HS to SO^'^ (HS" + 2O2 with the problems associated with high pH. Above > SO/" + H). Thiohacillns ferrooxidans is par- a pH of 8 or so, certain biomolecules, notably RNA, ticularly active in surface coal mining operations break down. Consequently, like acidophiles, alka- where exposure to oxygen of pyrite (FeS2) in the liphiles must maintain their cytopl coal seam triggers acid production from the meta- neutrality than their environment. Nevertheless, bolic activities of this and related bacteria. Runoff any proteins found in the cell wall or in the mem- from these habitats can often have a pH of less than brane that make contact with the environment must 2, fueling conditions for further acidophile activity. be stable to high pH. Indeed, many such enzymes The most acidophilic of all bacteria known thus have been studied and a number have found in- far is Picrophiliis oshimae^ whose pH optimum for dustrial applications, especially in the laundry de- growth is just 0.7 (Schleper et al., 1995) (Table 1). tergent industry. Detergents that are "enzyme en- Picrophilus oshimae is also a thermophile (temper- riched" contain proteases and lipases (enzymes that ature optimum, 60°C) so this organism must be sta- degrade proteins or fats, respectively, in clothing ble to both hot and acidic conditions. Cultures of stains) that function at the high pH of soapy solu- R. oshimae were isolated from an extremely acidic tions (Horikoshi & Grant, 1998). In addition, alkali-


Annals of the

Missouri Botanical Garden

active enzymes from thermophiles and psychro- molyte, the archaeon Haloharterium (Tahle 1) ron- philes have been discovered and commercialized to centrates large amounts of potassium (K^, as KCl) better target detergent additives to hot water or cold from its environment. Dissolv(Hl KCl in the cyto-

water applications, respectively.

plasm of Halobacterium cells is present at a con-

Besides keeping their cytoplasm near neutrality, centralion etjual to or slightly above that of the dis-

alkaliphiles have other biological problems to con- solved NaCl outside, and in this way c(41s maintain

tend with. For example, consider the problem of the tendency for water to enter and then^by prevent

membrane-mediated bioenergetics protons ex- dehydration. As would be expected from such a

traded to the external surface of the membrane en- salty cytoplasm, enzymes that function inside of

ter a sea of hydroxyl ions. Nevertheless, bio(;hem- cells of Ilalohacterium have evolved to require this

ical studies of this problem have shown that a large dose of K^ for catalytic activity, fiy contrast,

proton motive force is indeed formed by extreme membrane or cell wall-positioned prot<nns in Hal-

alkaliphiles and drives some of the energy-requir- obarterium require Na^ and are typically stable

ing reactions in the cell, such as motility and trans- only in the presence of high Na^ (Madigan & Orcn,

port. Sometimes in ATP synthesis, an ion gradient


F]xtreme halophiles are sourc(*s of a vari(»ty of

of Na^, rather than H^, drives this key bioenergetic

process in extreme alkaliphiles (Horikoshi & Grant, biomolecules that can function under salty condi-

1998). This is probably not surprising when one tions. Applications of salt-active enzymes include

considers that many (but not all) extreme alkali- those that can break down viscous materials pres-

phlles are also extreme halophiles (see below), re- ent in oil wells (oil is often found in geographic

quiring high salt as well as high pH for metabolism strata that contain salt) as well as enzymes tliat can

and reproduction.

cany out desirable transformations in highly salted


foods. In addition, some halophiles that produce organic compatible solutes have been commercial- ized for the production of these solutes as skin rare

Another remarkable group of extremophiles are supplements (Madigan & Orcn, 1999). the halophiles organisms adapted to grow best in

salty solutions (Oren, 1999; Ventosa et al., 1998). OlllFR ExTUKMOIMULls And for extreme halophiles like Halobacterium, a "salty solution" means anywhere from 25% NaCl

Extrt^mophilic microorganisms adapti^d to high up to saturation (32% NaCl) (Table 1). Halophilic pressure or which show no deleterious t^huts from microorganisms abound in hypersaline lakes such exposure to high levels of radiation are also known, as the Dead Sea, the Great Salt Lake, and solar salt Barophiles are microorganisms thai grow best under evaporation ponds. Such lakes are often colored red pressure greater than 1 atmosphere. Extreme bar- by the dense microbial communities of pigmented ophiles are the most interesting in this regard as

halophiles such as Halobacterium (Javor, 1989). they actually require pressure, and in some cases.

Other habitats for halophilic microorganisms in- extreme pressure, for growth (Table 1). Strain elude highly salted foods, saline soils, and under- MT41, for example, a bacterium isolated from ma- ground salt deposits. To date, a very large number rine sediments in the Mariana Trench near the Phil-

of halophilic bacteria have been grown in culture ippines (a depth of greater than 10,000 m), rccjuires