Enzymes in the Environment: Activity, Ecology and Applications - Chapter 13 doc

at the high end of the temperature scale, in the sense that most enzymes produced byhyperthermophiles also tend to be hyperthermophilic in their behavior, showing a tempera-ture optimum

13 Search for and Discovery of Microbial Enzymes from Thermally Extreme Environments in the Ocean

Jody W Deming and John A Baross

University of Washington, Seattle, Washington

The thermal end members of marine habitats on this planet are submarine mal systems (1), including the virtually unexplored subsurface biosphere beneath theirseafloor expressions (2–5), and subzero sea-ice systems (6), including their connections

hydrother-to permanently cold deep waters and sediments of polar regions (7) In considering thesethermally extreme environments, we build upon the axiom that extreme temperatures,especially the sharp thermal gradients they create in the ocean, have provided powerfulevolutionary forces to select for microbial enzymes with unique characteristics, unlikethose of their moderate temperature counterparts Inextricably related to the selective pres-sure of extreme temperature in these targeted environments are the elevated hydrostaticpressures at hydrothermal vents, which act to keep superheated fluids liquid in the deepsea (to temperatures above 400°C) (1), and the elevated salinities and other solutes in sea-ice formations, which act to keep supercooled fluids liquid even at the coldest of winter-

time temperatures (to ⫺35°C) (8,9) Just as organisms themselves must have suitableintracellular and membrane-bound enzymes to metabolize, replicate, and transcribe deoxy-ribonucleic acid (DNA) and grow under such combinations of extreme conditions, theextracellular transforming and degradative enzymes they release into their surroundingsmust be able to persist long enough to provide a useful nutritional return Although ourtendency to focus more on extracellular than intracellular enzymes in this chapter stemsfrom the more abundant information available on them and the applied interests in them(the two are linked), it is also rooted in an ecological appreciation of their importance tothe producing organisms in their natural settings (10).

II TERMINOLOGY

A The Microorganisms

Until recently, the search for microbial enzymes from extreme environments has invariablyinvolved the producing organisms themselves, either in laboratory culture or in their nativehabitats Terms used to describe the behavior of microorganisms at both ends of the tem-perature spectrum have undergone a series of revisions over the years, and sometimesapparent misuse, as the information base has increased and research emphases havechanged (11,12) Here, as elsewhere (1,11), we consider hyperthermophilic microorgan-isms as those that grow optimally at a temperature of 80°C or higher and to a maximaltemperature of at least 90°C; most are of marine origin, many of them isolated from thetype of hydrothermal vent systems that we consider here All are relatively new to science;they are differentiated from the more moderate thermophiles, known for decades, thathave maximal growth temperatures between 55°C and 80°C (thermophilic eukaryotes,algae and fungi, have maximal temperatures of 50°C to 60°C) At the lower end of thetemperature spectrum, we follow Morita’s (13) definition of psychrophilic microorganisms

as those that grow optimally at 15°C or lower and to a maximal temperature of 20°C.Psychrophiles are distributed worldwide in every type of cold environment, but those frommarine environments have been the object of study for nearly a century (14) Each ofthese definitions, for hyperthermophiles and for psychrophiles, clearly emphasizes optimalactivity at an extreme end of the temperature spectrum, coupled with the upper temperaturelimit for growth Consistent terms emphasizing the lower-temperature limit for growthare generally missing from the literature

The issue of a lower-temperature limit has contributed to some confusion in theliterature, especially in the realm of psychrophily, because of the many organisms capable

of growth (albeit slow) at near-freezing temperatures, even though they grow optimallyabove, and often well above, 20°C Depending on author and research perspective, suchorganisms have been called psychrotrophic, psychrotolerant, or facultatively psychrophilic(12,13,15) Blurring the picture further is the recent use of psychrophilic to mean anyorganism capable of growth at near-freezing temperatures, regardless of its growth opti-mum Some have argued that growth yield can be the more important variable and thatyield is not always linked to growth rate (16) Here we follow our oceanographic perspec-tives and use psychrotolerant to refer to those organisms that can grow at near-freezingtemperatures but most rapidly at approximate room temperature This choice parallels the(high-pressure) deep-sea literature, in which barotolerant refers to an organism that cangrow at elevated hydrostatic pressures but most rapidly at approximate room (atmospheric)

pressure (17) We reserve the use of psychrophilic to refer only to an organism optimized for growth at low temperatures (Toptⱕ 15°C and Tmax ⬍ 20°C) (13).

Less confusion in terminology figures in the high-temperature literature, since thesituation in the reverse direction does not appear to apply; that is, few if any organismswith optimal growth temperatures below 80°C can also grow at 90°C or higher As organ-isms with increasingly higher temperature optima and maxima for growth (as a generalrule, they increase in tandem) have been discovered, terms have easily accommodated the

new information, from thermophilic (Toptⱖ 55°C and Tmax⫽ 80°C) to hyperthermophilic (Topt ⱖ 80°C and Tmax ⱖ 90°C) to superthermophilic (Tmax ⱖ 115°C) (1,2) The term

extremely thermophilic, used somewhat loosely in the past, has generally been retired in favor of the defined term hyperthermophilic In the cold direction, the trend appears to

be from psychrophilic (Toptⱕ 15°C and Tmin⫽ 0°C) (13) to extremely psychrophilic (Toptⱕ

5°C and Tmin⫽ ⫺5°C); (18) to superpsychrophilic (Tmin⬍ ⫺5°C) (9)

at the high end of the temperature scale, in the sense that most enzymes produced byhyperthermophiles also tend to be hyperthermophilic in their behavior, showing a tempera-ture optimum for catalytic activity (Table 1) close to or greater than the Toptfor growth

of the organism The exceptions are almost always intracellular enzymes (four of fivecases shown in Table 1)

The greater potential for confusion again emerges at the low end of the temperaturescale Reference to an enzyme under study as psychrophilic rarely means that the enzymeitself expresses optimal activity at a temperature of 15°C or lower, since so few enzymes

with such a low Toptfor catalytic activity are known (Table 2) Depending on the tive of the investigator, reference to an enzyme as psychrophilic can mean that it wasproduced by a psychrophilic organism, produced by a psychrotolerant organism, active

perspec-at low temperperspec-atures (even if not optimally), or unstable perspec-at high temperperspec-atures (regardless

of its thermal activity optimum) Some papers report Toptfor catalytic activity but not fororganism growth, or vice versa, whereas others report maximal temperatures for enzymestability (or enzyme stability at a temperature selected for reasons of convenience, notnecessarily biological or ecological relevance) but not thermal optima (Table 2) We can

find no examples of an enzyme from a psychrophile that has a Toptfor activity lower thanthe growth optimum of the producing organism (Table 2), in contrast to the situation forhyperthermophiles (Table 1)

The most common terms in use for enzymes from psychrophilic (or psychrotolerant)

microorganisms are cold-active and cold-adapted, circumventing the terminology problem that stems from an emphasis on Topt for catalytic activity and focusing instead on theability of the enzyme to express significant activity at low temperatures, given a referencepoint for maximal activity at room temperature or higher In light of the still limitedinformation available on enzymes from psychrophiles (compared to hyperthermophiles),

we adopt a similar approach in this chapter, at the same time underscoring the prediction,

Table 1 Examples of Enzymes from Hyperthermophilic Heterotrophic Microorganisms, All Isolated from Marine Hydrothermal Vents, Ordered by Strain

(Toptfor Growth) and Thermal Activity Optimum

Enzyme

Organism (domain) Topt(°C) Enzyme, function Topt(°C) (time,°C)a References

(Bacteria)

β-Galactosidae, lactose hydrolysisb 80 N.A 79Hydrogenase, hydrogen production ⬎90 2 h @ 95 147, 148Xylanase A, xylan hydrolysisb 92 45 min @ 90 149Xylanase B, xylan hydrolysisb 105 3 h @ 90 149Glucose isomerase, glucose to fructose 105–110 10 min @ 120 73

Cellulase celA, cellulose hydrolysisb 95 N.A 85Xylanase, xylan hydrolysisb ⬎100 N.A 150α-Galactosidase, lactose hydrolysisb 100–103 9 h @ 85 84

2 h @ 90

3 min @ 100Cellulase celB, cellulose hydrolysisb 106 130 min @ 106 85

26 min @ 110

Thermococcus litoralis 85 DNA polymerase, DNA amplification 75 7 h @ 95 88, 151(Archaea)

Amylopullulanase, starch hydrolysisb 115 N.A 73

(Archaea)

Protease, peptide bond hydrolysis 85 N.A 153, 154Hydrogenase, hydrogen production 95 2 min @ 100 69, 147, 155α-Amylase, starch hydrolysisb 100 2 min @ 120 156, 157β-Glucosidase, cellobiose hydrolysisb 102–105 85 h @ 98 158Protease PfpI, peptide bond hydrolysis 105 N.A 159β-Mannosidase, mannan hydrolysisb 105 60 h @ 90 160Invertase, sucrose inversion 105 48 min @ 95 161α-Glucosidase, maltose hydrolysis 110 48 min @ 98 158, 162Serine protease, peptide bond hydrolysis 115 33 min @ 98 163Amylopullulanase, starch hydrolysisb 125 12 min @ 125 73

ES4 (Archaea)

a N.A., not available.

b Extracellular enzyme.

Table 2 Recent Examples of Enzymes from Psychrophilic Heterotrophic Marine Bacteria, Ordered by Strain (Environmental Source and ToptforGrowth) and Thermal Activity Optimum

Enzyme EnzymeGrowth activity half-lifeSource Organism Topt(°C) Enzyme Topt(°C) (time,°C) ReferencesSea ice, Antarctic Vibrio sp strain ANT300 7 Triosephosphate isomerase N.A.c 9 min @ 25 166

Phosphatasesd,e 17–23Proteasesd,e 28–30

Phosphatased,e 19Proteasesd,e 20—27α-Amylased,e 25Trypsind,e 30

Proteased,e 29

Sediments, Arctic Aquaspirillum arcticum 4 Malate dehydrogenase N.A 10 min @ 55 121

Seawater, Antarctic Pseudomonas aeruginosa N.A Proteasee ⬍25 2 min @ 45 23, 167

10 min @ 50Animal-associated, Psychrobacter immobilis ⬍10 β-Lactamasee 35 5 min @ 50 169,170

5 min @ 60

15 min @ 60

Deep sea Vibrio sp strain 5709 20 Proteasee 40 20 min @ 40 120

N.A., not available.

a Earlier work (e.g., 15,164,165), sometimes based on culture supernatants or partially purified protein preparations, reported similar thermal activity optima (in the range of 25 °C–

50 °C) and thermostabilities (e.g., 10 min at 40°C–70°C; 164).

bOnly the organisms listed from sea ice or sediments are strict psychrophiles (with both Toptfor growthⱕ15°C and Tmaxⱕ 20°C); those from polar seawater, polar animals, or the deep sea have been called psychrophilic by various investigators, but are not or may not be psychrophiles as defined here.

c Indications of optimal activity shifted to lower temperature or of pronounced heat lability.

d Preparation may have included multiple isozymes.

e Extracellular enzyme.

as supported by information from 1999 and 2000 (Table 2) (19,20), that new discoverieswill refocus attention on thermal activity optima that are indeed psychrophilic (ⱕ15°C).

In the realm of applications at either end of the temperature spectrum, however, neitheractivity optima nor thermal stability may be the essential enzyme feature: fidelity of ampli-fication (in the case of DNA polymerases) or novelty of chemical transformation maytake precedence Ultimately, an understanding of the balance between activity optima andthermal stability must be achieved Fortunately, this goal motivates much of the recentresearch on enzymes from both extremely hot and extremely cold marine environments

III BIOCHEMICAL CHALLENGES AT THERMAL EXTREMES

A Common and Divergent Themes

The ability of an organism to grow or survive at an extreme temperature poses specialphysiological and biochemical challenges Success depends upon both extrinsic and intrin-sic factors: elevated hydrostatic pressure or solute concentration at high temperatures (as

at deep-sea vents) and high salt or other solute concentration at low temperatures (as insea-ice brines) These can extend the permissive temperature range by their effects on theliquid state of water (and on other molecules); intrinsic factors associated with uniquelyevolved structural, catalytic, and informational macromolecules are essential The starkcontrast in levels of thermal energy inherent to very hot and very cold environments hasled to divergent growth and survival strategies for hyperthermophiles and psychrophiles

In the face of very high thermal energy in superheated fluids, the successful mophile maintains metabolic integrity and control by virtue of remarkably heat-stablemembranes, cell walls, and macromolecules, surviving supramaximal temperatures viaunique ‘‘heat-shock’’ proteins that stabilize macromolecules at otherwise denaturing tem-peratures (21,22) In the face of very low thermal energy in subzero fluids, the successfulpsychrophile must contend with greatly reduced rates of physical (e.g., diffusional), physi-ological, and biochemical processes, maintaining adequate membrane fluidity simply toacquire nutrition from its surroundings

hyperther-Compared to extrinsic factors involved in growth and survival strategies at thermalextremes or to the intrinsic factors of structural lipids and informational macromolecules,less is known about the vast array of enzymes required by an organism to be successful

at either end of the temperature spectrum Some basic properties that emerge from a parison of the extremes include that enzymes from psychrophiles have a lower free energy

com-of activation than enzymes from thermophiles (23), in keeping with the disparate levels

of thermal energy in their respective environments By definition, cold-adapted enzymeshave upper denaturation thresholds at relatively moderate temperatures (30°C–60°C) com-pared to hyperthermophilic enzymes, even though the same does not hold true in thereverse direction: only some hyperthermophilic enzymes are known to denature at coldtemperatures (near room temperature or below) (24); most remain stable as the temperaturedrops In spite of limited data, a relationship does appear to exist between the thermaloptima for enzyme activity and the half-life or thermostability of the enzyme at supraopti-

mal temperatures: the higher the Topt for activity the longer the lifetime at even highertemperatures (compare data for xylanases, for DNA polymerases, and for amylopullanases

inTable 1 and for proteases inTable 2)

Although analyses of the most basic features of enzymes—their amino acid quences—have yielded some insight into what makes an enzyme uniquely adapted to one

se-thermal extreme or the other, the combination of this information with other biochemicaland theoretical studies has been the most revealing (e.g., 25–27) For example, features

of a successful hyperthermophilic enzyme can include increased compactness, tion ofα helices, increased salt bridges and ion pairs for stabilizing secondary structures,

stabiliza-or an increased number of hydrogen bonds, each toward retaining stability in the face ofvery high denaturing temperatures The cold-adapted enzyme, in contrast, shows greaterflexibility and less compaction, lacks salt bridges and ion pairs, and has a reduced number

of hydrogen bonds, all toward retaining activity under very-low-energy near-freezing ditions No organism, however, appears to have evolved a uniform strategy for stabilizing

con-or allowing activity of all of its enzymes at a given extreme temperature Instead, its suite

of enzymes encompasses a range of unique combinations of molecular adapations thatreflect the host of complex evolutionary and ecological factors, including acquisition ofsuccessful traits through genetic exchange in the environment (28), that define a contempo-rary microorganism

A common theme for hyperthermophily and psychrophily, relating enzymes directly

to the producing organism (and thus allowing at least some common terminology), is that

the higher the Toptfor growth of the organism, the higher the Toptfor its enzymes: just asenzymes optimized for activity at the highest temperatures clearly derive from hyperther-mophiles adapted to growth at the highest temperatures (Table 1), enzymes with the lowestthermal optima derive from psychrophiles with the lowest growth optima (Table 2) Infact, all known cold-adapted enzymes express thermal activity optima that fall above the

Toptfor growth of the producing strain (Table 2) Although the same holds true in largepart for hyperthermophilic enzymes, some (mostly intracellular) enzymes are optimized

for activity below the Toptfor growth (Table 1), retaining only minimal stability lives of a few minutes) as the maximal temperature for growth is approached (29)

(half-B Intracellular Versus Extracellular Enzymes

If activity optima for enzymes, whether from hyperthermophiles or psychrophiles, areexamined according to general cellular location of the enzyme—intracellular (essential

to metabolism, DNA processing, growth) versus extracellular (typically hydrolytic

en-zymes that act independently of the organism)—the Toptfor extracellular enzymes almost

always falls above, and sometimes well above, the Toptfor growth (80% of the mophilic cases in Table 1; 100% of the psychrophilic cases in Table 2) Attempts tounderstand this locational discrepancy in thermal optima have been made by researchersstudying psychrophilic and psychrotolerant bacteria They have asked, What evolutionarypressure would select for extracellular enzymes optimized for activity at temperatures well

hyperther-above the Toptfor growth (25)? Would not an extracellular enzyme with greatest activity

at the Toptfor growth be ideal—or better yet, at the in situ temperature of the environment,which in the case of psychrophiles is invariably lower than its growth optimum? Whyshould extracellular enzymes have evolved differently in thermal properties than intracel-lular enzymes?

The biochemical processes underlying enzyme activity versus stability at a giventemperature have been proposed as a primary explanation for the phenomenon (16,23,25).Basically, an enzyme is least stable at the higher end of the temperature range over which

it is active For example, a psychrophile-derived extracellular enzyme optimized for ity at 30°C has a shorter half-life at that temperature than at lower ones It is thus morestable at the growth optimum for the organism (ⱕ15°C) and has its longest lifetime at

activ-the typical subzero temperatures encountered in a polar marine setting As long as activ-theenzyme retains enough activity at the lower temperatures, its longer lifetime can be seen

as a benefit to the producing organism What constitutes ‘‘enough’’ and ‘‘benefit’’ hasbeen explored in a quantitative model of microbial foraging by extracellular enzymesunder thermally moderate conditions (10); no similar quantitative analysis is available forthermal extremes (but see 9) The following consideration of enzyme foraging in light ofthe common physical features of our focal environments underscores the promise of thisstrategy for hyperthermophiles and psychrophiles and for a new generation of foragingmodels incorporating extreme temperatures

IV MICROBIAL FORAGING USING EXTRACELLULAR ENZYMES

A General Features

The quantitative modeling work of Vetter and associates (10) addresses the specific use

of extracellular enzymes as a bacterial foraging strategy in moderate-temperature vironments rich in particulate organic material (POM), of a size too large to pass the cellmembrane without extracellular hydrolysis The typical marine environment where thisstrategy is demonstrably advantageous involves an aggregation of POM-rich particles,either mineral grains with sorbed POM (as in marine sediments), or organic-rich detritalparticles (as in aggregates sinking through the water column) Adequate pore spacethrough which various solutes, from enzymes to POM hydrolysate, can diffuse is alsoessential (Fig 1) Although not yet considered in a modeling context for their specialfeatures, both hydrothermal structures and sea ice are rich in interior colonizable surfaces,often laden with organic material in patches, and can be highly porous They representideal settings for the use of extracellular enzymes as a foraging strategy and for recognitionand improved dissection of the consequences of evolutionary pressure on enzyme adapta-tion at extreme temperatures

microen-B Foraging in Hot Sulfide Structures

Actively venting sulfide structures on the seafloor are, by definition, composed of mineralgrains, of variable composition depending on local chemical and thermal conditions forprecipitation and deposition (30) They are colonized in their cooler portions by animalsthat produce organic, especially chitinous, structures and polymers that remain after theorganism has sought new territory or succumbed to either a predator or thermochemicalchange in the habitat The sulfide formations are clearly porous, often functioning as visi-ble diffusers releasing cooled hydrothermal fluids into the surrounding seawater Theirinterior portions are known to support abundant heterotrophic (and other) microbial popu-lations (1,31,32), zoned phylogenetically (Bacteria versus Archaea;Fig 2) according topermissive temperatures The prediction from this combination of features alone is thatPOM foraging using extracellular enzymes is an important strategy for the growth andsurvival of heterotrophic microorganisms living within these structures The additional factthat known hyperthermophilic heterotrophs release a wide variety of highly thermostableenzymes into culture media in the laboratory (Table 1) makes seafloor sulfide structuresobvious sites for future exploration and discovery of new enzymes, especially extracellularhydrolytic enzymes Although no direct environmental searches of enzyme activity inhydrothermal structures have yet been made to our knowledge, such an approach could

Figure 1 Schematic diagram of an aggregate of particles with an immobile enzyme-releasingbacterium at the center The aggregate environment is composed of impermeable inorganic grains(shaded shapes), patches of organic material (black shapes, too large to cross the cell membrane)sorbed to the grains, and seawater-filled (or brine-filled, in sea ice) spaces through which solutesdiffuse Heavy arrows represent enzyme diffusing away from the organism; dashed arrows, hydroly-sate diffusing away from the organic material, where it is produced enzymatically (Modified fromRef 10.)

prove fruitful, just as new microorganisms continue to emerge from direct examination

of seafloor hydrothermal sites and effluents from the subsurface biosphere (5,32,33).Special features to consider in the search for hyperthermophilic enzymes (or organ-isms) or in predictive modeling efforts in advance are the sharp temperature gradients,established across distances that measure in centimeters, in actively venting sulfide struc-tures (Fig 2); the other thermally linked gradients in chemical parameters, from pH to

Eh (oxidation status) to a multitude of dissolved inorganic and organic species (1,28); andthe influence of advection versus diffusion through pore spaces Since hydrostatic pressurefigures importantly at deep-sea hydrothermal vents, acting to keep superheated fluids inthe liquid phase and to select for barophilic and barotolerant microorganisms (2), it mustalso be considered in the study of microbial enzymes from these environments (28,29,34).The critical feature of the lifetime of a given enzyme, especially when sorbed to mineralgrains within an actively flushing vent structure, determines not only its detectability andutility as a foraging tool to the producing organism and its neighbors (10), perhaps inzones too hot to permit the organism itself (28), but also its performance immobilizedunder extreme conditions and thus its attraction to biotechnologists

Figure 2 Schematic diagrams of examples of the hottest (seafloor hydrothermal sulfide structure)and coldest (wintertime Arctic sea ice) marine habitats, depicting common physical features of inte-rior colonizable surface area, fluid-filled pore space, and sharp thermal gradients Note that bacterialand archaeal zonations have been explored in sulfide structures (modified from Ref 28), but notyet in sea ice (modified from Ref 45), whereas fine-scale temperature and chemical properties ofpore fluids are better known for sea ice (e.g., salinity gradients parallel thermal gradients inversely)than sulfide structures.

C Foraging in Subzero Sea Ice

The three basic features of the enzyme foraging model of Vetter and coworkers (10) forparticle aggregates (Fig 1) also pertain to the other end of the temperature spectrum formicrobial life and enzymatic activity epitomized by sea ice Aggregates of mineral grainsand other particles and precipitates (including microorganisms and salts) are known toconcentrate within the fluid inclusions of sea ice (6), most notably in the Arctic, whereseabed sediments entrain into coastal ice as it forms (35) These aggregates include POM-rich detrital particles (36) and large exopolymers (37) as a result of the autotrophic andheterotrophic communities that develop annually within the ice cover (38–40), as well asgenerally elevated levels of dissolved organic carbon (41,42) including enzymes (19,20).The sea-ice matrix is also highly porous, especially in summertime, flushing regularlywith the tides or influence of waves while retaining particle aggregates and organismswithin it (43,44) Even during wintertime (in the Arctic), when sea-ice temperatures candrop below⫺20°C (Fig 2) to as low as⫺35°C, depending on snow cover and atmosphericconditions (8), interior movements of brine fluid through finely connected channels arepossible on a scale relevant to bacteria and enzymes This has been demonstrated byphysical analyses of undisturbed ice sections using nuclear magnetic resonance (NMR)and transmission microscopy (45)

In contrast to research on hydrothermal structures, less information is available onthe abundance or possible zonation, phylogenetic or otherwise (Fig 2), of microorganisms

in these coldest of wintertime sea-ice habitats (e.g., 18, 36) Only in 1999 was a structive (nonwarming, nonmelting) method for studying microbial life in supercooledice developed (36) Although extreme temperatures determine the solid phase of bothhydrothermal structures (by controlling mineral precipitation reactions) and sea ice (byfreezing water), only the hydrothermal structure remains intact for ready study at tempera-tures less extreme than those in situ Sea-ice structure changes nonuniformly with everyincremental change (up or down) in temperature, presenting special challenges to apostsampling evaluation of in situ microbial communities, products, or processes.Nevertheless, the prediction from the three basic features (abundant attachment sites,organic material, and porosity) that enzyme foraging is an important microbial strategyfor growth and survival in sea ice has been supported by direct environmental measure-ments in both wintertime (18) and summertime sea-ice samples (19,20) Not only havehydrolytic activities on substrate analogs for protein, chitin, and various carbohydratesbeen readily detected, but, where measured and compared across other subzero environ-ments (Arctic seawater and sinking aggregates), the lowest thermal optima for enzymeactivities were observed in multiyear sea ice (19) The optima were consistently psychro-philic, down to 10°C, compared to previous reports of 30°C–50°C (19, 20, and citationstherein) (Table 2) In other words, the ice cover over the Arctic Ocean, which in someareas persists through a decade of winters (rarely if ever the case in Antarctic waters),clearly selects for cold-adapted and even strictly psychrophilic enzymes, as it does forpsychrophilic organisms (discussed later), making it an obvious environment for continuedsearch and discovery of new enzymes in this thermal class

nonde-Special features to consider in a search for cold-adapted enzymes in sea ice resemblethose for seafloor sulfide structures, albeit at subzero temperatures: sharp thermal gradients

in wintertime ice (Fig 2), linked salinity (and other chemical) gradients (Fig 2), and theinfluence of advection versus diffusion Elevated salinities, as well as concentrations ofother solutes, are key to depressing the freezing point and maintaining fluid-filled pore

spaces In fact, physiological studies of sea-ice bacteria suggest that salinity (and pH)gradients may be as critical to potential microbial succession and zonation with the ice-brine matrix as the cardinal growth parameter of temperature (46) Unique to the cold end

of the temperature spectrum exemplified by sea ice are the problems of increased viscosity

as temperature drops and salinity rises (in wintertime) and of interior vertical mixing withtidal and wave action (in summertime) The lifetime of an extracellular enzyme sorbed

to particle surfaces in situ under extremely cold, saline, and viscous conditions is as tant a factor to the organisms in sea ice (and to those who study them) as it is in the case

impor-of extremely hot, chemically reduced, and nonviscous conditions within hydrothermalvent structures

Indeed, enzyme lifetime or stability emerges as a critical factor in scenarios thathelp to explain why extracellular enzymes appear optimized for activity at temperatureswell above what they experience in their natural settings, whether very hot or very cold

In an early ecological scenario for subzero marine sediments (47), absent specific tion on enzyme activity relative to lifetime, both characteristics were assumed to be re-stricted at in situ temperature, such that luxury or excessive production of extracellularenzymes was invoked to account for sufficient hydrolytic return to support the microbialcommunity clearly present in the environment Indeed, some psychrophiles have sincebeen shown in the laboratory to produce maximal amounts of extracellular enzymes atsuboptimal growth temperatures (48), where they also appear to require elevated concen-trations of dissolved organic matter for activity (49,50) However, we also now understandthat an enzyme optimized for activity at a temperature well above the growth optimumfor the producing organism (and thus the in situ temperature, in the case of psychrophiles)

informa-is more stable at that lower growth (or in situ) temperature than at its own Toptfor activity(see 16, 23, 25 for discussion at the biochemical–molecular level as to why a cold-adaptedenzyme is relatively unstable in its optimally active state) The balance between activityand stability at the environmentally relevant temperature is thus also understood to deter-mine the extracellular enzyme of greatest benefit to its producer (9,10,12,16,19,20,23,25,51) We suggest that evolutionary pressure on microorganisms to feed competitivelyand thus survive in microenvironments rich in POM (10) (Fig 1), but at temperaturessuboptimal for growth, has selected for the production of extracellular enzymes with abalance between cell-free activity and lifetime that favors longevity at the in situ tempera-ture and thus long-term return of hydrolysate to the organism and its neighbors In contrast,membrane-bound and intracellular enzymes (which remain under cellular control and thuscan be recycled and produced anew, as needed) with maximal catalytic activity at a giventemperature help define that temperature as the optimum for growth

This enzyme-based foraging and growth scenario is so far exemplified by a

psychro-philic bacterium, Colwellia sp strain 34H, enriched from near-freezing Arctic sediments

(7) and later shown to be optimized for growth at 5°C–8°C (20) The organism producesextracellular proteases with unusually low thermal optima for activity (20°C) (Table 2)

A crude preparation of these proteases from mass cultivation of the organism, one thatstill includes some intracellular proteases, expresses a lower thermal optimum for activity(13°C) (Fig 3), as would be predicted for such an enzyme mix The fraction of enzymeactivity remaining in this preparation after a holding period at the environmentally relevanttemperature of 0°C was significantly greater than that remaining at warmer temperaturesmore permissive of both growth and enzyme activity (Fig 3) In other words, to surviveand even grow in a permanently cold environment, an organism is well served both byextracellular enzymes adapted for maximal activity at temperatures well above in situ (and

Figure 3 Activity of a crude preparation of proteases, specifically leucine-aminopeptidase

(LA-Pase) activity from Colwellia sp strain 34H (19) relative to maximum hydrolytic rate (R/Rmax) overtime at different incubation temperatures Note LAPase activity decreasing to greater extent at highertemperatures (e.g., 59% loss after 76 h at 13°C, where loss is attributed to enzyme denaturation)than lower temperatures (e.g., 33% loss after 76 h at 0°C), as well as the shift in activity optimumfrom 13°C to 10°C to 8°C with increasing holding time Solid lines, cubic spline curve fits; errorbars, 95% confidence intervals for triplicate measurements at each temperature (From A Hustonand J Deming, unpublished.)

optimal growth) temperature, and thus well-designed at the molecular level for a longlifetime of activity (albeit at modest catalytic rate) at the environmentally relevant tempera-ture, and by intracellular enzymes more closely optimized to in situ conditions and thusfor metabolism and growth A similar scenario, one not yet discussed in the literature,can be proposed for hyperthermophilic enzymes and their producing organisms, oftenfound at growth-permissive temperatures along sharp thermal gradients within seafloorsulfide structures (Fig 2) Some surprises in the hyperthermophilic scenario may be instore, however, given the fact that some intracellular enzymes from hyperthermophiles(e.g., DNA polymerases) express thermal activity optima below growth optima of theproducing strains yet also retain significant activity even at extremely high temperatures(Table 1)

D Future Foraging Scenarios

For both ends of the temperature spectrum, the described enzyme-based foraging andgrowth scenarios and new ones yet to be developed benefit from rigorous and innovativeanalysis of the characteristics of both intracellular and extracellular enzymes purified frommodel organisms; the research and technology communities may benefit from discovery

of new enzymes or features of enzyme activity and thermostability in the process Heeding

an ecological perspective should continue to be useful, given that thermal optima apparent

to researchers in short-term incubation experiments in the laboratory may not be

synony-mous with thermal optima relevant to an organism dependent (52) on a flux of dissolvedcompounds from enzymes already released and functioning over longer periods in theenvironment For example, note the shift in thermal activity optima for cold-adapted prote-ases from 13°C to 10°C to 8°C as a function of increasing holding time (Fig 3) We alsosuggest that the further exploratory study of microbial enzymes produced in environmentscharacterized by sharp thermal gradients may yield enzymes with both high catalytic activ-ity and long lifetimes at extreme temperatures (hot or cold), a combination of featuresthat so far has been observed only as a result of genetic engineering (described later) andapparently not of evolutionary pressures in nature The temporally and spatially fluctuatingthermal gradients within sulfide structures and sea ice may have provided the necessaryselective pressure.

V STATUS OF THE SEARCH FOR HYPERTHERMOPHILIC MICROORGANISMS AND ENZYMES

A Focus on Culturable Hyperthermophiles

Although the discovery of hyperthermophilic microorganisms at marine hydrothermalvents was reported in 1982 (53,54), their potentially exciting activities in situ have beenstudied by few and remain poorly constrained (1, 28) The in situ activities of enzymesthat hyperthermophiles may release into their surroundings are completely unknown Thisgeneral lack of ecological information on the functioning of either hyperthermophilic or-ganisms or enzymes in their natural settings stands in contrast to what is known aboutorganisms and enzymes at the other end of the temperature spectrum (see Sec V B);marine psychrophiles have been known and studied for almost a century, much of thework ecologically motivated from the outset (14) Perhaps because of the immediate recog-nition of practical applications for new organisms functional at ever higher temperatures(11,55), research efforts now ongoing worldwide have focused heavily on organism andenzyme performance under controlled laboratory conditions, with specific biotechnologi-cal or industrial goals motivating the choice of organism, enzyme, or test conditions Thedesire to achieve a fundamental understanding of the biochemical, metabolic, and geneticbasis for hyperthermophily has often been presented as a better means to manipulate strainsand their products in vitro for commercial purposes However, the first whole-genomesequence for any organism, information of the most fundamental nature, was obtained for

the deep-sea hyperthermophile Methanococcus jannaschii (56).

Although ecological considerations beg study and enzyme foraging scenarios forhyperthermophiles have not yet been formulated, the acquisition of culturable hyperther-mophiles from marine hydrothermal vents now borders on routine Current repositories

of marine hyperthermophiles, virtually all of which are obligately anaerobic, include sentatives of 25 genera (examples of which are shown inFig 4in italics) and physiologicalprocesses as diverse as methanogenesis, iron oxidization, other forms of chemoautotrophy,sulfate reduction, and other forms of heterotrophy With the exception of methanogenicgenera, which contain a wide range of thermal classes of methanogens, all of these generacontain only hyperthermophilic organisms As species have been added over time, culturecollections have become dominated by heterotrophic hyperthermophiles in a limited num-ber of genera of both Archaea and Bacteria These organisms typically require complexorganic compounds, including peptides or carbohydrates, to meet their carbon demands(21,57) For the initially exciting pace of discovery of new and diverse hyperthermophilic

repre-Figure 4 Universal phylogenetic tree based on 16S rRNA sequences, showing the three domains

of Bacteria, Archaea, and Eukarya and featuring the hyperthermophilic genera (in italics), whichfall within the prokaryotic domains of Bacteria and Archaea Cultured psychrophiles fall within thebacterial divisions of Proteobacteria, Flavobacteria, and Gram-positive bacteria (seeFig 5); themarine Crenarchaeota comprise a large group of uncultured presumptive psychrophiles within theArchaea Distances were derived from numbers of mutations; the root, from sequences of the twosubunits of the F1-ATPases and the translation elongation factors EF-1α (Tu) and EF-2 (G) (Modi-fied from Ref 65.)

organisms to continue, however, innovative sampling and culturing approaches beyondthe now-standard heterotrophic sulfur-based media must be pursued The continuing prom-ise of discovery is evidenced by sampling strategies that access, at the seafloor, recentfluid emissions from the subsurface biosphere (2,4) and by inventive culturing media thatyield new organisms and even the potential for novel metabolisms (33) For example, anorganism isolated in 2000 from a seafloor eruption site on the Gorda Ridge in the NortheastPacific Ocean likely represents a new genus and can function metabolically as a hetero-troph, autotroph, and iron reducer (33)

Direct molecular approaches to assessing microbial diversity underscore the mense and still untapped diversity of hyperthermophiles Analyses of small-subunit ribo-nucleic acid (RNA) sequences in environmental samples from terrestrial hot springs firstrevealed this untapped diversity in both domains of Bacteria and Archaea (58,59) In anearly similar analysis of a submarine vent environment, specifically a sample of microbialmat from Loihi Seamount (60), sequence analyses of fewer than 50 clones appeared toreflect the untapped diversity in that habitat adequately Subsequent analyses of the type

im-of sulfidic structures that we have targeted here, with their sharp thermal gradients andhigher end member values, have revealed a much greater degree of diversity, such thatanalysis of hundreds of clones can still be inadequate to the task (31,32,61) The presence

of so many potentially unique and hyperthermophilic organisms necessarily indicates anequally untapped diversity of hyperthermophilic enzymes, awaiting discovery

In the meantime, culture collections have provided rich depths to plumb: several

well-studied heterotrophic hyperthermophiles have become the targets of concentratedsearches for enzymes with unusual thermal or other properties Of all of the known genera

of hyperthermophiles, only three heterotrophic ones—Pyrococcus and Thermococcus of the Archaea and Thermotoga of the Bacteria—have yielded species that can be grown

reproducibly to high cell densities in the laboratory, making them model organisms forenzyme studies in vitro In fact, most of the physiological and enzymological studies havebeen carried out with three species (Table 1), Pyrococcus furiosus (62), Thermococcus litoralis (63), and Thermotoga maritima (64).

By far the largest number of hyperthermophilic species, and most of the ized hyperthermophilic proteins, belong to the archaeal family of Thermococcales of thekingdom Euryarchaeota (65) (Fig 4) This family is cosmopolitan in that its membershave been isolated from all hydrothermal environments sampled so far All are known toutilize carbohydrates by a glycolytic pathway that includes some unusual tungsten-con-taining enzymes (24,66); most have a growth requirement for amino acids and peptides,and for elemental sulfur (21) Tungsten concentrations, as well as levels of a wide variety

character-of other metals, can be very high in portions character-of seafloor sulfide structures deposited atextreme temperatures (1,67), raising interesting evolutionary (and biotechnological) ques-tions about metal-based enzymes and proteins in general External sources of amino acids,proteins, and other organic compounds for hyperthermophiles in their native settings havebeen hypothesized (1), but not yet confirmed quantitatively

For the two genera, Pyrococcus and Thermococcus, that constitute the family of

Thermococcales, approximately 40 species have been described They are routinely lated from near-vent sites on the seafloor, from samples of originally hot sulfidic rocksand other structures, from alvinellid worms that colonize actively venting structures, andoccasionally from samples of hot fluid emerging from such structures (1) Not only arethese hyperthermophiles easy to grow, they are also hardy, surviving storage under low-temperature oxic conditions (68; J Baross, unpublished observations) Their ability to uti-lize a wide range of organic substrates is reflected in their production of a diverse array

iso-of hydrolytic enzymes (55,69,70) (Table 1); the oxidoreductases and dehydrogenases volved in their fermentative metabolisms are the enzymes with metal (tungsten) centers(24) Both the diversity and the properties of high-temperature enzymes can vary signifi-cantly among similar species (69) Enough information is now available to recognize thatfor a single organism the thermal properties of enzymes in the same functional class can

in-also vary significantly (see proteases for Pyrococcus furiosus in Table 1) The latter finding

opens new evolutionary and ecological scenarios for hyperthermophiles, e.g., the hood that thermal gradients fluctuating in time and space in the vent environment, andperhaps especially in the subsurface biosphere (1,28,33), may have selected for suites ofisozymes that make an organism uniquely adapted to survive, and possibly grow, across

likeli-a wider rlikeli-ange of conditions thlikeli-an previously envisioned

B Focus on Commercially Important Enzymes

Apart from enzyme studies of some of the metalloproteins involved in hyperthermophilic

metabolism mentioned, most of the studies of the best-known species of Pyrococcus and Thermococcus have focused on enzymes of commercial importance Classes of hydrolytic

enzymes have received particular emphasis; catalytic activity at high temperatures andextreme thermostability are frequently the properties of greatest interest Hydrolytic en-zymes are used by organisms to degrade peptides, complex carbohydrates, and lipids to