13 SearchforandDiscoveryofMicrobial EnzymesfromThermallyExtreme EnvironmentsintheOcean JodyW.DemingandJohnA.Baross UniversityofWashington,Seattle,Washington I.INTRODUCTION Thevastarrayoforganicandinorganiccompoundsproducedinanddeliveredtotheocean makeitahavenforanextraordinarydiversityofmicroorganismsthatuseadiversearray ofcatalyticagentsinthesynthesis,transformation,anddegradationofthesematerials. Themicrobialproductionofenzymesfunctionalatmoderatethermalconditionsinthe marineenvironmentiswellknown(seeChapter4).Herewearguefromobservations, experimental results, and hypothetical scenarios that thermally extreme environments in the ocean offer new and continuing vistas for discovering enzymes of unique metabolic, ecological, and evolutionary significance, as well as for creating practical applications in the realms of biotechnology and bioremediation. We explore the physical features and, perhaps surprising, commonalities of very hot and very cold marine habitats, as well as the enzymes and producing microorganisms already known from these environments, to develop predictions of enzymes awaiting discovery. The potential for application of geno- mics, proteomics, and other forms of genetic access and manipulation as new search and discovery tools that can be made even more powerful with ecological insight is high- lighted. The thermal end members of marine habitats on this planet are submarine hydrother- mal systems (1), including the virtually unexplored subsurface biosphere beneath their seafloor expressions (2–5), and subzero sea-ice systems (6), including their connections to permanently cold deep waters and sediments of polar regions (7). In considering these thermally extreme environments, we build upon the axiom that extreme temperatures, especially the sharp thermal gradients they create in the ocean, have provided powerful evolutionary forces to select for microbial enzymes with unique characteristics, unlike those of their moderate temperature counterparts. Inextricably related to the selective pres- sure of extreme temperature in these targeted environments are the elevated hydrostatic pressures at hydrothermal vents, which act to keep superheated fluids liquid in the deep sea (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- Copyright © 2002 Marcel Dekker, Inc. time temperatures (to Ϫ35°C) (8,9). Just as organisms themselves must have suitable intracellular and membrane-bound enzymes to metabolize, replicate, and transcribe deoxy- ribonucleic acid (DNA) and grow under such combinations of extreme conditions, the extracellular transforming and degradative enzymes they release into their surroundings must be able to persist long enough to provide a useful nutritional return. Although our tendency to focus more on extracellular than intracellular enzymes in this chapter stems from 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 to the producing organisms in their natural settings (10). II. TERMINOLOGY A. The Microorganisms Until recently, the search for microbial enzymes from extreme environments has invariably involved the producing organisms themselves, either in laboratory culture or in their native habitats. 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 sometimes apparent misuse, as the information base has increased and research emphases have changed (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 maximal temperature of at least 90°C; most are of marine origin, many of them isolated from the type 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, that have maximal growth temperatures between 55°C and 80°C (thermophilic eukaryotes, algae and fungi, have maximal temperatures of 50°Cto60°C). At the lower end of the temperature 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 from marine environments have been the object of study for nearly a century (14). Each of these definitions, for hyperthermophiles and for psychrophiles, clearly emphasizes optimal activity at an extreme end of the temperature spectrum, coupled with the upper temperature limit for growth. Consistent terms emphasizing the lower-temperature limit for growth are generally missing from the literature. The issue of a lower-temperature limit has contributed to some confusion in the literature, especially in the realm of psychrophily, because of the many organisms capable of growth (albeit slow) at near-freezing temperatures, even though they grow optimally above, and often well above, 20°C. Depending on author and research perspective, such organisms have been called psychrotrophic, psychrotolerant, or facultatively psychrophilic (12,13,15). Blurring the picture further is the recent use of psychrophilic to mean any organism 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 that yield 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-freezing temperatures but most rapidly at approximate room temperature. This choice parallels the (high-pressure) deep-sea literature, in which barotolerant refers to an organism that can grow at elevated hydrostatic pressures but most rapidly at approximate room (atmospheric) Copyright © 2002 Marcel Dekker, Inc. pressure(17).Wereservetheuseofpsychrophilictoreferonlytoanorganismoptimized forgrowthatlowtemperatures(T opt Յ15°CandT max Ͻ20°C)(13). Lessconfusioninterminologyfiguresinthehigh-temperatureliterature,sincethe situationinthereversedirectiondoesnotappeartoapply;thatis,fewifanyorganisms withoptimalgrowthtemperaturesbelow80°Ccanalsogrowat90°Corhigher.Asorgan- ismswithincreasinglyhighertemperatureoptimaandmaximaforgrowth(asageneral rule,theyincreaseintandem)havebeendiscovered,termshaveeasilyaccommodatedthe newinformation,fromthermophilic(T opt Ն55°CandT max ϭ80°C)tohyperthermophilic (T opt Ն80°CandT max Ն90°C)tosuperthermophilic(T max Ն115°C)(1,2).Theterm extremelythermophilic,usedsomewhatlooselyinthepast,hasgenerallybeenretiredin favorofthedefinedtermhyperthermophilic.Inthecolddirection,thetrendappearsto befrompsychrophilic(T opt Յ15°CandT min ϭ0°C)(13)toextremelypsychrophilic(T opt Յ 5°CandT min ϭϪ5°C);(18)tosuperpsychrophilic(T min ϽϪ5°C)(9). B.TheEnzymes Auniformapproachtoclassifyingenzymesonasimilarthermalbasissofarhaseluded theresearchcommunity.Acommonapproachhasbeentorefertotheenzymeaccording tothegrowthoptimumoftheorganismthatproducesit,ratherthanaparticularthermal featureoftheenzymeitself.Enzymesproducedbyhyperthermophilicorganismshave thusbeencalledhyperthermophilicenzymes,whereasthoseproducedbypsychrophilic organismshavebeencalledpsychrophilicenzymes.Thisapproachworksreasonablywell atthehighendofthetemperaturescale,inthesensethatmostenzymesproducedby hyperthermophilesalsotendtobehyperthermophilicintheirbehavior,showingatempera- tureoptimumforcatalyticactivity(Table1)closetoorgreaterthantheT opt forgrowth oftheorganism.Theexceptionsarealmostalwaysintracellularenzymes(fouroffive casesshowninTable1). Thegreaterpotentialforconfusionagainemergesatthelowendofthetemperature scale.Referencetoanenzymeunderstudyaspsychrophilicrarelymeansthattheenzyme itselfexpressesoptimalactivityatatemperatureof15°Corlower,sincesofewenzymes withsuchalowT opt forcatalyticactivityareknown(Table2).Dependingontheperspec- tive of the investigator, reference to an enzyme as psychrophilic can mean that it was produced by a psychrophilic organism, produced by a psychrotolerant organism, active at low temperatures (even if not optimally), or unstable at high temperatures (regardless of its thermal activity optimum). Some papers report T opt for catalytic activity but not for organism growth, or vice versa, whereas others report maximal temperatures for enzyme stability (or enzyme stability at a temperature selected for reasons of convenience, not necessarily biological or ecological relevance) but not thermal optima (Table 2). We can find no examples of an enzyme from a psychrophile that has a T opt for activity lower than the growth optimum of the producing organism (Table 2), in contrast to the situation for hyperthermophiles (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 T opt for catalytic activity and focusing instead on the ability of the enzyme to express significant activity at low temperatures, given a reference point for maximal activity at room temperature or higher. In light of the still limited information available on enzymes from psychrophiles (compared to hyperthermophiles), we adopt a similar approach in this chapter, at the same time underscoring the prediction, Copyright © 2002 Marcel Dekker, Inc. Table 1 Examples of Enzymes from Hyperthermophilic Heterotrophic Microorganisms, All Isolated from Marine Hydrothermal Vents, Ordered by Strain (T opt for Growth) and Thermal Activity Optimum Enzyme Growth activity Enzyme half-life Organism (domain) T opt (°C) Enzyme, function T opt (°C) (time, °C) a References Thermotoga maritima 80 4-α-Glucano-transferase, starch hydrolysis b 70 3 h @ 80 81 (Bacteria) β-Galactosidae, lactose hydrolysis b 80 N.A. 79 Hydrogenase, hydrogen production Ͼ90 2 h @ 95 147, 148 Xylanase A, xylan hydrolysis b 92 45 min @ 90 149 Xylanase B, xylan hydrolysis b 105 3 h @ 90 149 Glucose isomerase, glucose to fructose 105–110 10 min @ 120 73 Thermotoga neopolitana 80 Mannanase, mannan hydrolysis b 91 13 h @ 90 84 (Bacteria) 35 min @ 100 Cellulase celA, cellulose hydrolysis b 95 N.A. 85 Xylanase, xylan hydrolysis b Ͼ100 N.A. 150 α-Galactosidase, lactose hydrolysis b 100–103 9 h @ 85 84 2h@90 3 min @ 100 Cellulase celB, cellulose hydrolysis b 106 130 min @ 106 85 26 min @ 110 Copyright © 2002 Marcel Dekker, Inc. Thermococcus litoralis 85 DNA polymerase, DNA amplification 75 7 h @ 95 88, 151 (Archaea) Amylopullulanase, starch hydrolysis b 115 N.A. 73 Pyrococcus furiosus 100 DNA polymerase, DNA amplification Ͼ75 20 h @ 95 92, 152 (Archaea) Protease, peptide bond hydrolysis 85 N.A. 153, 154 Hydrogenase, hydrogen production 95 2 min @ 100 69, 147, 155 α-Amylase, starch hydrolysis b 100 2 min @ 120 156, 157 β-Glucosidase, cellobiose hydrolysis b 102–105 85 h @ 98 158 Protease PfpI, peptide bond hydrolysis 105 N.A. 159 β-Mannosidase, mannan hydrolysis b 105 60 h @ 90 160 Invertase, sucrose inversion 105 48 min @ 95 161 α-Glucosidase, maltose hydrolysis 110 48 min @ 98 158, 162 Serine protease, peptide bond hydrolysis 115 33 min @ 98 163 Amylopullulanase, starch hydrolysis b 125 12 min @ 125 73 Pyrococcus sp. strain 100 Amylopullulanase, starch hydrolysis b 118 10 min @ 120 74 ES4 (Archaea) a N.A., not available. b Extracellular enzyme. Copyright © 2002 Marcel Dekker, Inc. Table 2 Recent a Examples of Enzymes from Psychrophilic b Heterotrophic Marine Bacteria, Ordered by Strain (Environmental Source and T opt for Growth) and Thermal Activity Optimum Enzyme Enzyme Growth activity half-life Source Organism T opt (°C) Enzyme T opt (°C) (time, °C) References Sea ice, Antarctic Vibrio sp. strain ANT300 7 Triosephosphate isomerase N.A. c 9 min @ 25 166 Colwellia demingiae strains ACAM607, IC169 10–12 Trypsins d,e 12–14 N.A. c 105 Phosphatases d,e 17–23 Proteases d,e 28–30 Cytophaga-like sp. strains IC164, IC166 N.A. β-Galactosidase d,e 15 N.A. c 105 Phosphatase d,e 19 Proteases d,e 20—27 α-Amylase d,e 25 Trypsin d,e 30 Pseudoalteromonas sp. strain IC000 N.A. Trypsin d,e 22 N.A. c 105 Protease d,e 29 Shewanella gelidimarina strain ACAM456 N.A. β-Galactosidase d,e 24 N.A. c 105 Copyright © 2002 Marcel Dekker, Inc. Sediments, Arctic Aquaspirillum arcticum 4 Malate dehydrogenase N.A. c 10 min @ 55 121 Colwellia sp. strain 34H 5–8 Protease d,e 20 N.A. c 20 Seawater, Antarctic Pseudomonas aeruginosa N.A. Protease e Ͻ25 2 min @ 45 23, 167 Alteromonas haloplanctis Ͻ15 α-Amylase e 27 10 min @ 50 16, 168 Bacillus sp. strains TA39, TA41 N.A. Subtilisin e 45 90 min @ 25 16, 130 10 min @ 50 Animal-associated, Psychrobacter immobilis Ͻ10 β-Lactamase e 35 5 min @ 50 169,170 Antarctic (or N.A.) Lipase e N.A. N.A. c 171 Shewanella sp. N.A. Phosphatase 30 10 min @ 50 172 5 min @ 60 Flavobacterium balustinum strains P104–107 10–20 Proteases e 30–40 30 min @ 20 173 15 min @ 60 Flavobacterium sp. N.A. β-Mannanase e 35 N.A. c 174 Deep sea Vibrio sp. strain 5709 20 Protease e 40 20 min @ 40 120 Vibrio sp. strain 5710 N.A. Malate dehydrogenase N.A. c N.A. c 140 Photobacterium sp. strain SS9 N.A. Malate dehydrogenase N.A. N.A. 175 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). b Only the organisms listed from sea ice or sediments are strict psychrophiles (with both T opt for growth Յ15°C and T max Յ 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. Copyright © 2002 Marcel Dekker, Inc. assupportedbyinformationfrom1999and2000(Table2)(19,20),thatnewdiscoveries willrefocusattentiononthermalactivityoptimathatareindeedpsychrophilic(Յ15°C). Intherealmofapplicationsateitherendofthetemperaturespectrum,however,neither activityoptimanorthermalstabilitymaybetheessentialenzymefeature:fidelityofampli- fication(inthecaseofDNApolymerases)ornoveltyofchemicaltransformationmay takeprecedence.Ultimately,anunderstandingofthebalancebetweenactivityoptimaand thermalstabilitymustbeachieved.Fortunately,thisgoalmotivatesmuchoftherecent researchonenzymesfrombothextremelyhotandextremelycoldmarineenvironments. III.BIOCHEMICALCHALLENGESATTHERMALEXTREMES A.CommonandDivergentThemes Theabilityofanorganismtogroworsurviveatanextremetemperatureposesspecial physiologicalandbiochemicalchallenges.Successdependsuponbothextrinsicandintrin- sicfactors:elevatedhydrostaticpressureorsoluteconcentrationathightemperatures(as atdeep-seavents)andhighsaltorothersoluteconcentrationatlowtemperatures(asin sea-icebrines).Thesecanextendthepermissivetemperaturerangebytheireffectsonthe liquidstateofwater(andonothermolecules);intrinsicfactorsassociatedwithuniquely evolvedstructural,catalytic,andinformationalmacromoleculesareessential.Thestark contrastinlevelsofthermalenergyinherenttoveryhotandverycoldenvironmentshas ledtodivergentgrowthandsurvivalstrategiesforhyperthermophilesandpsychrophiles. Inthefaceofveryhighthermalenergyinsuperheatedfluids,thesuccessfulhyperther- mophilemaintainsmetabolicintegrityandcontrolbyvirtueofremarkablyheat-stable membranes,cellwalls,andmacromolecules,survivingsupramaximaltemperaturesvia unique‘‘heat-shock’’proteinsthatstabilizemacromoleculesatotherwisedenaturingtem- peratures(21,22).Inthefaceofverylowthermalenergyinsubzerofluids,thesuccessful psychrophilemustcontendwithgreatlyreducedratesofphysical(e.g.,diffusional),physi- ological,andbiochemicalprocesses,maintainingadequatemembranefluiditysimplyto acquirenutritionfromitssurroundings. Comparedtoextrinsicfactorsinvolvedingrowthandsurvivalstrategiesatthermal extremesortotheintrinsicfactorsofstructurallipidsandinformationalmacromolecules, lessisknownaboutthevastarrayofenzymesrequiredbyanorganismtobesuccessful ateitherendofthetemperaturespectrum.Somebasicpropertiesthatemergefromacom- parisonoftheextremesincludethatenzymesfrompsychrophileshavealowerfreeenergy ofactivationthanenzymesfromthermophiles(23),inkeepingwiththedisparatelevels ofthermalenergyintheirrespectiveenvironments.Bydefinition,cold-adaptedenzymes haveupperdenaturationthresholdsatrelativelymoderatetemperatures(30°C–60°C)com- paredtohyperthermophilicenzymes,eventhoughthesamedoesnotholdtrueinthe reversedirection:onlysomehyperthermophilicenzymesareknowntodenatureatcold temperatures(nearroomtemperatureorbelow)(24);mostremainstableasthetemperature drops.Inspiteoflimiteddata,arelationshipdoesappeartoexistbetweenthethermal optimaforenzymeactivityandthehalf-lifeorthermostabilityoftheenzymeatsupraopti- maltemperatures:thehighertheT opt foractivitythelongerthelifetimeatevenhigher temperatures(comparedataforxylanases,forDNApolymerases,andforamylopullanases inTable1andforproteasesinTable2). Although analyses of the most basic features of enzymes—their amino acid se- quences—have yielded some insight into what makes an enzyme uniquely adapted to one Copyright © 2002 Marcel Dekker, Inc. thermalextremeortheother,thecombinationofthisinformationwithotherbiochemical andtheoreticalstudieshasbeenthemostrevealing(e.g.,25–27).Forexample,features ofasuccessfulhyperthermophilicenzymecanincludeincreasedcompactness,stabiliza- tionofαhelices,increasedsaltbridgesandionpairsforstabilizingsecondarystructures, oranincreasednumberofhydrogenbonds,eachtowardretainingstabilityinthefaceof veryhighdenaturingtemperatures.Thecold-adaptedenzyme,incontrast,showsgreater flexibilityandlesscompaction,lackssaltbridgesandionpairs,andhasareducednumber ofhydrogenbonds,alltowardretainingactivityundervery-low-energynear-freezingcon- ditions.Noorganism,however,appearstohaveevolvedauniformstrategyforstabilizing orallowingactivityofallofitsenzymesatagivenextremetemperature.Instead,itssuite ofenzymesencompassesarangeofuniquecombinationsofmolecularadapationsthat reflectthehostofcomplexevolutionaryandecologicalfactors,includingacquisitionof successfultraitsthroughgeneticexchangeintheenvironment(28),thatdefineacontempo- rarymicroorganism. Acommonthemeforhyperthermophilyandpsychrophily,relatingenzymesdirectly totheproducingorganism(andthusallowingatleastsomecommonterminology),isthat thehighertheT opt forgrowthoftheorganism,thehighertheT opt foritsenzymes:justas enzymesoptimizedforactivityatthehighesttemperaturesclearlyderivefromhyperther- mophilesadaptedtogrowthatthehighesttemperatures(Table1),enzymeswiththelowest thermaloptimaderivefrompsychrophileswiththelowestgrowthoptima(Table2).In fact, all known cold-adapted enzymes express thermal activity optima that fall above the T opt for growth of the producing strain (Table 2). Although the same holds true in large part for hyperthermophilic enzymes, some (mostly intracellular) enzymes are optimized for activity below the T opt for growth (Table 1), retaining only minimal stability (half- lives of a few minutes) as the maximal temperature for growth is approached (29). B. Intracellular Versus Extracellular Enzymes If activity optima for enzymes, whether from hyperthermophiles or psychrophiles, are examined 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 T opt for extracellular enzymes almost always falls above, and sometimes well above, the T opt for growth (80% of the hyperther- mophilic cases in Table 1; 100% of the psychrophilic cases in Table 2). Attempts to understand this locational discrepancy in thermal optima have been made by researchers studying psychrophilic and psychrotolerant bacteria. They have asked, What evolutionary pressure would select for extracellular enzymes optimized for activity at temperatures well above the T opt for growth (25)? Would not an extracellular enzyme with greatest activity at the T opt for 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? Why should extracellular enzymes have evolved differently in thermal properties than intracel- lular enzymes? The biochemical processes underlying enzyme activity versus stability at a given temperature 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 activ- ity at 30°C has a shorter half-life at that temperature than at lower ones. It is thus more stable at the growth optimum for the organism (Յ15°C) and has its longest lifetime at Copyright © 2002 Marcel Dekker, Inc. thetypicalsubzerotemperaturesencounteredinapolarmarinesetting.Aslongasthe enzymeretainsenoughactivityatthelowertemperatures,itslongerlifetimecanbeseen asabenefittotheproducingorganism.Whatconstitutes‘‘enough’’and‘‘benefit’’has beenexploredinaquantitativemodelofmicrobialforagingbyextracellularenzymes underthermallymoderateconditions(10);nosimilarquantitativeanalysisisavailablefor thermalextremes(butsee9).Thefollowingconsiderationofenzymeforaginginlightof thecommonphysicalfeaturesofourfocalenvironmentsunderscoresthepromiseofthis strategyforhyperthermophilesandpsychrophilesandforanewgenerationofforaging modelsincorporatingextremetemperatures. IV.MICROBIALFORAGINGUSINGEXTRACELLULARENZYMES A.GeneralFeatures ThequantitativemodelingworkofVetterandassociates(10)addressesthespecificuse ofextracellularenzymesasabacterialforagingstrategyinmoderate-temperaturemicroen- vironmentsrichinparticulateorganicmaterial(POM),ofasizetoolargetopassthecell membranewithoutextracellularhydrolysis.Thetypicalmarineenvironmentwherethis strategyisdemonstrablyadvantageousinvolvesanaggregationofPOM-richparticles, eithermineralgrainswithsorbedPOM(asinmarinesediments),ororganic-richdetrital particles(asinaggregatessinkingthroughthewatercolumn).Adequateporespace throughwhichvarioussolutes,fromenzymestoPOMhydrolysate,candiffuseisalso essential(Fig.1).Althoughnotyetconsideredinamodelingcontextfortheirspecial features,bothhydrothermalstructuresandseaicearerichininteriorcolonizablesurfaces, oftenladenwithorganicmaterialinpatches,andcanbehighlyporous.Theyrepresent idealsettingsfortheuseofextracellularenzymesasaforagingstrategyandforrecognition andimproveddissectionoftheconsequencesofevolutionarypressureonenzymeadapta- tionatextremetemperatures. B.ForaginginHotSulfideStructures Activelyventingsulfidestructuresontheseafloorare,bydefinition,composedofmineral grains,ofvariablecompositiondependingonlocalchemicalandthermalconditionsfor precipitationanddeposition(30).Theyarecolonizedintheircoolerportionsbyanimals thatproduceorganic,especiallychitinous,structuresandpolymersthatremainafterthe organismhassoughtnewterritoryorsuccumbedtoeitherapredatororthermochemical changeinthehabitat.Thesulfideformationsareclearlyporous,oftenfunctioningasvisi- blediffusersreleasingcooledhydrothermalfluidsintothesurroundingseawater.Their interiorportionsareknowntosupportabundantheterotrophic(andother)microbialpopu- lations(1,31,32),zonedphylogenetically(BacteriaversusArchaea;Fig.2)accordingto permissivetemperatures.Thepredictionfromthiscombinationoffeaturesaloneisthat POMforagingusingextracellularenzymesisanimportantstrategyforthegrowthand survivalofheterotrophicmicroorganismslivingwithinthesestructures.Theadditionalfact thatknownhyperthermophilicheterotrophsreleaseawidevarietyofhighlythermostable enzymesintoculturemediainthelaboratory(Table1)makesseafloorsulfidestructures obvious sites for future exploration and discovery of new enzymes, especially extracellular hydrolytic enzymes. Although no direct environmental searches of enzyme activity in hydrothermal structures have yet been made to our knowledge, such an approach could Copyright © 2002 Marcel Dekker, Inc. [...]... wintertime ice (Fig 2), linked salinity (and other chemical) gradients (Fig 2), and the in uence of advection versus diffusion Elevated salinities, as well as concentrations of other solutes, are key to depressing the freezing point and maintaining fluid-filled pore Copyright © 2002 Marcel Dekker, Inc spaces In fact, physiological studies of sea-ice bacteria suggest that salinity (and pH) gradients may... mutations; the root, from sequences of the two subunits of the F1-ATPases and the translation elongation factors EF-1α (Tu) and EF-2 (G) (Modified from Ref 65.) organisms to continue, however, innovative sampling and culturing approaches beyond the now-standard heterotrophic sulfur-based media must be pursued The continuing promise of discovery is evidenced by sampling strategies that access, at the seafloor,... HYPERTHERMOPHILIC MICROORGANISMS AND ENZYMES A Focus on Culturable Hyperthermophiles Although the discovery of hyperthermophilic microorganisms at marine hydrothermal vents was reported in 1982 (53,54), their potentially exciting activities in situ have been studied by few and remain poorly constrained (1, 28) The in situ activities of enzymes that hyperthermophiles may release into their surroundings... unconserved region, and near the substrate binding area) In another directed evolution study (129), both the thermostability (at high temperatures) and the activity (at low temperatures) of the cold-adapted protease subtilisin S41 from the Antarctic Bacillus strain TA41 (130 ) were improved substantially and simultaneously, i.e., in the same mutant strain Similar approaches using random mutagenesis have... (41,42) including enzymes (19,20) The sea-ice matrix is also highly porous, especially in summertime, flushing regularly with the tides or in uence of waves while retaining particle aggregates and organisms within it (43,44) Even during wintertime (in the Arctic), when sea-ice temperatures can drop below Ϫ20°C (Fig 2) to as low as Ϫ35°C, depending on snow cover and atmospheric conditions (8), interior... live within thermal gradients and close promixity to temperatures higher than their apparent (at atmospheric pressure) maximum for growth and survival (Fig 2) Much work remains to be done VI STATUS OF THE SEARCH FOR PSYCHROPHILIC MICROORGANISMS AND ENZYMES A Focus on Organisms and Enzymes in Their Native Habitats In contrast to the study of hyperthermophiles and their enzymes, in which commercial interests... ecological information on the functioning of either hyperthermophilic organisms or enzymes in their natural settings stands in contrast to what is known about organisms 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 the work ecologically motivated from the outset (14) Perhaps because of the immediate... successful in improving thermal stability and pH Copyright © 2002 Marcel Dekker, Inc tolerance or modifying catalytic activity and substrate specificity of enzymes performing at the high end of the temperature spectrum (131 134 ) VII CONCLUSIONS Whether initially motivated by applied interests, ecological issues, or a desire to understand the fundamental basis for thermal adaptations, researchers exploring thermally... ascribed to incorrect folding or assembly of the protein (138 ) For example, the half-life of native glutamate dehydrogenase from Pyrococcus furiosus is 10 h at 100°C, whereas the half-life of the recombinant protein measures only in minutes at the same temperature (135 ) In order to take full advantage of the potential for discovery of novel enzymes from genome sequences of either hyperthermophiles... buried in hot sulfide structures during their formation, and by polychaetes that directly inhabit sulfide structures (1) The combination of Thermococcus chitinophagous or Pyrococcus furiosus as the organism, chitin as the target POM (Fig 1), chitinases (or other glycosyl hydrolases) as the foraging tools, and sulfide structures as the environmental setting may provide an ideal start for an enzyme foraging . Inc. mouswiththermaloptimarelevanttoanorganismdependent(52)onafluxofdissolved compoundsfromenzymesalreadyreleasedandfunctioningoverlongerperiodsinthe environment.Forexample,notetheshiftinthermalactivityoptimaforcold-adaptedprote- asesfrom13°Cto10°Cto8°Casafunctionofincreasingholdingtime(Fig.3).Wealso suggestthatthefurtherexploratorystudyofmicrobialenzymesproducedinenvironments characterizedbysharpthermalgradientsmayyieldenzymeswithbothhighcatalyticactiv- ityandlonglifetimesatextremetemperatures(hotorcold),acombinationoffeatures thatsofarhasbeenobservedonlyasaresultofgeneticengineering(describedlater )and apparentlynotofevolutionarypressuresinnature.Thetemporallyandspatiallyfluctuating thermalgradientswithinsulfidestructuresandseaicemayhaveprovidedthenecessary selectivepressure. V.STATUSOFTHESEARCHFORHYPERTHERMOPHILIC MICROORGANISMSANDENZYMES A.FocusonCulturableHyperthermophiles Althoughthediscoveryofhyperthermophilicmicroorganismsatmarinehydrothermal ventswasreportedin1982(53,54),theirpotentiallyexcitingactivitiesinsituhavebeen studiedbyfewandremainpoorlyconstrained(1,28).Theinsituactivitiesofenzymes thathyperthermophilesmayreleaseintotheirsurroundingsarecompletelyunknown.This generallackofecologicalinformationonthefunctioningofeitherhyperthermophilicor- ganismsorenzymesintheirnaturalsettingsstandsincontrasttowhatisknownabout organismsandenzymesattheotherendofthetemperaturespectrum(seeSec.V.B); marinepsychrophileshavebeenknownandstudiedforalmostacentury,muchofthe workecologicallymotivatedfromtheoutset(14).Perhapsbecauseoftheimmediaterecog- nitionofpracticalapplicationsforneworganismsfunctionalateverhighertemperatures (11,55),researcheffortsnowongoingworldwidehavefocusedheavilyonorganismand enzymeperformanceundercontrolledlaboratoryconditions,withspecificbiotechnologi- calorindustrialgoalsmotivatingthechoiceoforganism,enzyme,ortestconditions .The desiretoachieveafundamentalunderstandingofthebiochemical,metabolic,andgenetic basisforhyperthermophilyhasoftenbeenpresentedasabettermeanstomanipulatestrains andtheirproductsinvitroforcommercialpurposes.However ,the rstwhole-genome sequenceforanyorganism,informationofthemostfundamentalnature,wasobtainedfor thedeep-seahyperthermophileMethanococcusjannaschii(56). Althoughecologicalconsiderationsbegstudyandenzymeforagingscenariosfor hyperthermophileshavenotyetbeenformulated,theacquisitionofculturablehyperther- mophilesfrommarinehydrothermalventsnowbordersonroutine.Currentrepositories ofmarinehyperthermophiles,virtuallyallofwhichareobligatelyanaerobic,includerepre- sentativesof25genera(examplesofwhichareshowninFig.4initalics)andphysiological processes. Inc. low-molecular-weightcomponentsthatareeasilytransportedintothecell.Thebeststudied ofthehyperthermophilichydrolyticenzymesaretheproteasesandtheglycosylhydrolases, knowntoberemarkablythermostable(Table1)andtoshowveryhighratesofactivity attheirthermaloptima.Theirfunctionsforthecelldiffer,however,withcellularlocation. Somehyperthermophilicproteaseshavebeenreportedasintracellular,performingregula- toryandcatabolicfunctions,includingdegradinginactiveproteinsoractivatingothers; othershavebeenreportedasperiplasmicormembrane-associated,involvedindegrading peptidesfornutritionandgrowth.Thosehydrolyticenzymesusedextracellularlyfigure importantlyinthedevelopmentofenzymeforagingscenariosforhyperthermophiles.Dif- ferentlylocatedproteaseshavebeenstudiedfromPyrococcusfuriosus:theirthermalactiv- ityoptimarangefrom85°Cto115°C(Table1)(70),butwithnoobviousrelationship betweenT opt andintra-orextracellularlocation.ComparedtoEscherichiacoli,however, fromwhich36proteaseshavebeenidentified(71),onlyalimitednumberofproteases havebeenobtainedandstudiedfromhyperthermophilic(orpsychrophilic)organisms. Theglycosylhydrolasesfromhyperthermophiles,whichactoncomplexcarbohy- drates,arethemostthermallystableenzymesyetcharacterized.WhenP.furiosusandT. litoralis,normallyculturedinheterotrophicmediacontainingpeptides,aregrownonmalt- oseinstead,theyreachhighcellyieldsandproduceanextremelythermostableα-glucosi- dasewithanactivityoptimumof108°Candahalf-lifeof48hat98°C(72)(Table1) .The amylopullanasesfromPyrococcusspeciesES4andP.furiosushavethemostthermophilic enzymesyetdescribed.Inhydrolyzingboththe -1 , 4and -1 ,6linkagesinstarch,they showactivityattemperaturesabove125°C,wheretheyarestabilizedbytheadditionof Ca 2ϩ (73,74).Glycosylhydrolasesingeneralmaythusmeetthestartingpremisefora successfulextracellularforagerwhentheinsituenvironmentaltemperaturefallsator belowtheoptimalgrowthtemperatureoftheproducingstrain(e.g.,at100°Corbelow inasulfidestructure):theirthermalactivityoptimaaremuchhigher,implyinggreater longevityattheinsitutemperature.Evenwhereenzymelifetimeislimited,atextreme pointsinthethermalgradientspanningasulfidestructure(Fig.2),beneficialenzymic ‘‘work’’fortheproducingorganismoritsneighborsispossible.Utilizablehydrolysate maybeproducedandreturnedviadiffusiontomilderlocationsalongthegradientwhere theorganismsreside. Perhapsoneofthemoreecologicallyinterestingdiscoveriesaboutglycosylhy- drolasesfromhyperthermophilesisthatThermococcuschitinophagus,anarchaealhyper- thermophilefromdeep-seahydrothermalvents,canhydrolyzechitin(75).Subsequent analysisofthegenomesequenceforPyrococcusfuriosusrevealedthepresenceoftwo chitinases(aswellasothercarbohydrate-hydrolyzingenzymes),eventhoughthisorgan- ismisnotknowntodegradechitininculture(76).Fewerglycosylhydrolasesingeneral havebeenfoundinthePyrococcushorikoshiigenome(77),andonlyoneglycosylhy- drolasewasidentifiedfromthegenomeofthearchaealhyperthermophileArchaeglobus fulgidus(78).Theabundanceandcharacteristicsofchitinasesandotherglycosylhy- drolasesinhyperthermophilicArchaeaclearlyvarywithspecies;thatvariationinturn mayberelatedtotheirecologicalniche.Themajorsourcesofchitinforhyperthermophiles insubmarinehydrothermalventenvironmentsarethetubesconstructedbyvestimentiferan tubeworms,whichareknowntobecomeburiedinhotsulfidestructuresduringtheirforma- tion,andbypolychaetesthatdirectlyinhabitsulfidestructures(1).Thecombinationof ThermococcuschitinophagousorPyrococcusfuriosusastheorganism,chitinasthetarget POM(Fig.1),chitinases(orotherglycosylhydrolases)astheforagingtools,andsulfide structures. extracellular than intracellular enzymes in this chapter stems from the more abundant information available on them and the applied interests in them (the two are linked), it is also rooted in an ecological