... Inc.mouswiththermaloptimarelevanttoanorganismdependent(52)onafluxofdissolvedcompoundsfromenzymesalreadyreleasedandfunctioningoverlongerperiodsintheenvironment.Forexample,notetheshiftinthermalactivityoptimaforcold-adaptedprote-asesfrom13°Cto10°Cto8°Casafunctionofincreasingholdingtime(Fig.3).Wealsosuggestthatthefurtherexploratorystudyofmicrobialenzymesproducedinenvironmentscharacterizedbysharpthermalgradientsmayyieldenzymeswithbothhighcatalyticactiv-ityandlonglifetimesatextremetemperatures(hotorcold),acombinationoffeaturesthatsofarhasbeenobservedonlyasaresultofgeneticengineering(describedlater )and apparentlynotofevolutionarypressuresinnature.Thetemporallyandspatiallyfluctuatingthermalgradientswithinsulfidestructuresandseaicemayhaveprovidedthenecessaryselectivepressure.V.STATUSOFTHESEARCHFORHYPERTHERMOPHILICMICROORGANISMSANDENZYMESA.FocusonCulturableHyperthermophilesAlthoughthediscoveryofhyperthermophilicmicroorganismsatmarinehydrothermalventswasreportedin1982(53,54),theirpotentiallyexcitingactivitiesinsituhavebeenstudiedbyfewandremainpoorlyconstrained(1,28).Theinsituactivitiesofenzymesthathyperthermophilesmayreleaseintotheirsurroundingsarecompletelyunknown.Thisgenerallackofecologicalinformationonthefunctioningofeitherhyperthermophilicor-ganismsorenzymesintheirnaturalsettingsstandsincontrasttowhatisknownaboutorganismsandenzymesattheotherendofthetemperaturespectrum(seeSec.V.B);marinepsychrophileshavebeenknownandstudiedforalmostacentury,muchoftheworkecologicallymotivatedfromtheoutset(14).Perhapsbecauseoftheimmediaterecog-nitionofpracticalapplicationsforneworganismsfunctionalateverhighertemperatures(11,55),researcheffortsnowongoingworldwidehavefocusedheavilyonorganismandenzymeperformanceundercontrolledlaboratoryconditions,withspecificbiotechnologi-calorindustrialgoalsmotivatingthechoiceoforganism,enzyme,ortestconditions .The desiretoachieveafundamentalunderstandingofthebiochemical,metabolic,andgeneticbasisforhyperthermophilyhasoftenbeenpresentedasabettermeanstomanipulatestrainsandtheirproductsinvitroforcommercialpurposes.However ,the rstwhole-genomesequenceforanyorganism,informationofthemostfundamentalnature,wasobtainedforthedeep-seahyperthermophileMethanococcusjannaschii(56).Althoughecologicalconsiderationsbegstudyandenzymeforagingscenariosforhyperthermophileshavenotyetbeenformulated,theacquisitionofculturablehyperther-mophilesfrommarinehydrothermalventsnowbordersonroutine.Currentrepositoriesofmarinehyperthermophiles,virtuallyallofwhichareobligatelyanaerobic,includerepre-sentativesof25genera(examplesofwhichareshowninFig.4initalics)andphysiologicalprocesses ... Inc.thermalextremeortheother,thecombinationofthisinformationwithotherbiochemicalandtheoreticalstudieshasbeenthemostrevealing(e.g.,25–27).Forexample,featuresofasuccessfulhyperthermophilicenzymecanincludeincreasedcompactness,stabiliza-tionofαhelices,increasedsaltbridgesandionpairsforstabilizingsecondarystructures,oranincreasednumberofhydrogenbonds,eachtowardretainingstabilityinthefaceofveryhighdenaturingtemperatures.Thecold-adaptedenzyme,incontrast,showsgreaterflexibilityandlesscompaction,lackssaltbridgesandionpairs,andhasareducednumberofhydrogenbonds,alltowardretainingactivityundervery-low-energynear-freezingcon-ditions.Noorganism,however,appearstohaveevolvedauniformstrategyforstabilizingorallowingactivityofallofitsenzymesatagivenextremetemperature.Instead,itssuiteofenzymesencompassesarangeofuniquecombinationsofmolecularadapationsthatreflectthehostofcomplexevolutionaryandecologicalfactors,includingacquisitionofsuccessfultraitsthroughgeneticexchangeintheenvironment(28),thatdefineacontempo-rarymicroorganism.Acommonthemeforhyperthermophilyandpsychrophily,relatingenzymesdirectlytotheproducingorganism(andthusallowingatleastsomecommonterminology),isthatthehighertheToptforgrowthoftheorganism,thehighertheToptforitsenzymes:justasenzymesoptimizedforactivityatthehighesttemperaturesclearlyderivefromhyperther-mophilesadaptedtogrowthatthehighesttemperatures(Table1),enzymeswiththelowestthermaloptimaderivefrompsychrophileswiththelowestgrowthoptima(Table2) .In fact, ... Inc.C.ForaginginSubzeroSeaIceThethreebasicfeaturesoftheenzymeforagingmodelofVetterandcoworkers(10)forparticleaggregates(Fig.1)alsopertaintotheotherendofthetemperaturespectrumformicrobiallifeandenzymaticactivityepitomizedbyseaice.Aggregatesofmineralgrainsandotherparticlesandprecipitates(includingmicroorganismsandsalts)areknowntoconcentratewithinthefluidinclusionsofseaice(6),mostnotablyintheArctic,whereseabedsedimentsentrainintocoastaliceasitforms(35).TheseaggregatesincludePOM-richdetritalparticles(36)andlargeexopolymers(37)asaresultoftheautotrophicandheterotrophiccommunitiesthatdevelopannuallywithintheicecover(38–40),aswellasgenerallyelevatedlevelsofdissolvedorganiccarbon(41,42)includingenzymes(19 ,20) .Thesea-icematrixisalsohighlyporous,especiallyinsummertime,flushingregularlywiththetidesorinfluenceofwaveswhileretainingparticleaggregatesandorganismswithinit(43,44).Evenduringwintertime(intheArctic),whensea-icetemperaturescandropbelow 20 C(Fig.2)toaslowasϪ35°C,dependingonsnowcoverandatmosphericconditions(8),interiormovementsofbrinefluidthroughfinelyconnectedchannelsarepossibleonascalerelevanttobacteriaandenzymes.Thishasbeendemonstratedbyphysicalanalysesofundisturbedicesectionsusingnuclearmagneticresonance(NMR)andtransmissionmicroscopy(45).Incontrasttoresearchonhydrothermalstructures,lessinformationisavailableontheabundanceorpossiblezonation,phylogeneticorotherwise(Fig.2),ofmicroorganismsinthesecoldestofwintertimesea-icehabitats(e.g.,18,36).Onlyin1999wasanonde-structive(nonwarming,nonmelting)methodforstudyingmicrobiallifeinsupercooledicedeveloped(36).Althoughextremetemperaturesdeterminethesolidphaseofbothhydrothermalstructures(bycontrollingmineralprecipitationreactions)andseaice(byfreezingwater),onlythehydrothermalstructureremainsintactforreadystudyattempera-tureslessextremethanthoseinsitu.Sea-icestructurechangesnonuniformlywitheveryincrementalchange(upordown)intemperature,presentingspecialchallengestoapostsamplingevaluationofinsitumicrobialcommunities,products,orprocesses.Nevertheless,thepredictionfromthethreebasicfeatures(abundantattachmentsites,organicmaterial,andporosity)thatenzymeforagingisanimportantmicrobialstrategyforgrowthandsurvivalinseaicehasbeensupportedbydirectenvironmentalmeasure-mentsinbothwintertime(18)andsummertimesea-icesamples(19 ,20) .Notonlyhavehydrolyticactivitiesonsubstrateanalogsforprotein,chitin,andvariouscarbohydratesbeenreadilydetected,but,wheremeasuredandcomparedacrossothersubzeroenviron-ments(Arcticseawaterandsinkingaggregates),thelowestthermaloptimaforenzymeactivitieswereobservedinmultiyearseaice(19).Theoptimawereconsistentlypsychro-philic,downto10°C,comparedtopreviousreportsof30°C–50°C(19 ,20, andcitationstherein)(Table2).Inotherwords,theicecoverovertheArcticOcean,whichinsomeareaspersiststhroughadecadeofwinters(rarelyifeverthecaseinAntarcticwaters),clearlyselectsforcold-adaptedandevenstrictlypsychrophilicenzymes,asitdoesforpsychrophilicorganisms(discussedlater),makingitanobviousenvironmentforcontinuedsearchanddiscoveryofnewenzymesinthisthermalclass.Specialfeaturestoconsiderinasearchforcold-adaptedenzymesinseaiceresemblethoseforseafloorsulfidestructures,albeitatsubzerotemperatures:sharpthermalgradientsinwintertimeice(Fig.2),linkedsalinity(andotherchemical)gradients(Fig.2),andthe in uence...