Extremophiles in Deep-Sea Environments PDF
Preview Extremophiles in Deep-Sea Environments
Springer J apan KK K. Horikoshi, K. Tsujii (Eds.) Extremophiles in Deep-Sea Environments With 122 Figures Springer Koki Horikoshi Director General The DEEPSTAR Group Japan Marine Science and Technology Center (JAMSTEC) 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan Kaoru Tsujii TeamLeader The DEEPSTAR Group Japan Marine Science and Technology Center (JAMSTEC) 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan ISBN 978-4-431-68008-6 Library of Congress Cataloging-in-Publication Data Extremophiles in deep-sea environments I K. Horikoshi, K. Tsujii (eds.). p. cm. Includes bibliographical references. ISBN 978-4-431-68008-6 ISBN 978-4-431-67925-7 (eBook) DOI 10.1007/978-4-431-67925-7 1. Extreme environments-Microbiology. 2. Benthos-Microbiology. 3. Submarine trenches. I. Horikoshi, Koki. II. Tsujii, Kaoru. QRlOO.9 .E96 1999 579'.1777-dc21 99-36537 Printed on acid-free paper © Springer Japan 1999 Originally published by Springer Verlag Tokyo Berlin Heidelberg New York in 1999 Softcover reprint of the hardcover 1s t edition 1999 This work is subject to copyright. AII rights are reserved whether the whole or part of the material is concerned, specifical1y the rights of translation, reprinting, reuse of ilIustrations, recitation, broadcast ing, reproduction on microfilms or in other ways, and storage in data banks. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Moriyama, Japan SPIN: 10728618 Preface OnFebruary 28, 1996,Japanese researchers working on the DEEPSTARprogram at the Japan Marine Science and Technology Center (JAMSTEC) broke their own world record by sending the unmanned submersible Kaiko (the Japanese word for "trench")to 10898metersbeneaththePacificOcean. Kaikosentbackvideoimages oflifein thedepths oftheMarianaTrench, thedeepest pointin the world'soceans. AbriefbutexcitedfaxfromthemothershipdescribedthescenecapturedonKaiko's camera:'ThebedoftheMarianaTrenchwasfilledwithafinemudofreddishbrownish particles. There were norocks orcracksatall and itresembledadesert. However, very unusual organisms were observed here and there." In the following fax, they describedtypesofseaurchin,jellyfishmovingfairly fast, and theexcrementofsea organisms. My colleagues also saw a fast-moving shrimp about three centimeters long. Therewasalsoakind ofseacucumber,which was thesamesizeas thejelly fish. Kaikoscoopedoutsamplesofmud to bringtothesurface. Now we would be able to isolatelivingcreaturesfrom thedeepestpointoftheMarianaTrench. Notsomanyyearsago,almostall biologistsbelievedthatlifecouldsurviveonly within so-called moderate environments; so when microbiologists looked around for interesting bacteria and other life-forms, they attempted to isolate microorgan isms only from moderateenvironments. About30yearsago, Iwas visiting Florence, Italy. Iwas lookingat the Renais sance buildings, which were very differentfrom Japanese architecture. About500 yearsago,noJapanesecouldpossiblyhaveimaginedthisRenaissanceculture. "There mightbeawhole new worldofmicroorganisms indifferent unexploredcultures," I thought."Couldtherebeanentirelyunknowndomainofmicroorganismsatalkaline pH?" The acidic environment was being studied, probably because most food is acidic. However, hardlyany work had been done in the alkalineregion. Science,justas much as the arts, relies upon asense ofromance and intuition. Upon my return to Japan, I prepared an alkaline medium containing I% sodium v VI Preface carbonate,placedasmallamountofsoilinit,andincubateditovernightat37°C. To mysurprise,variousmicroorganismsflourished inall 30testtubes. Herewasanew alkaline world which was utterly different from the neutral world discovered by Pasteur. I named these microorganisms which thrive in alkalineenvironments"al kaliphiles," and conducted systematic microbial physiological studies ofthem. At the same time I focused my intereston theenzymes produced by thesealkaliphilic microorganisms as well as on their molecular genetics. The results showed that thesemicroorganisms,whicharecompletelydifferentfromanypreviouslyreported, werewidelydistributedontheglobeandthey producednewproducts.This wasmy first encounter with a kind ofextremophile and led to my subsequent work with alkaliphiles. Thefive-yearSuperbugProjectoftheJapaneseERATOprogramwaslaunched in 1984tosearchforextremophilesthatgrowinextremeenvironmentsand totry to usetheiruniquepropertiestoestablisha"NewBiotechnology." Thisresearchproject resulted inmanysignificantscientificdiscoveries. TheSuperbugProjectwasarea sonablysuccessfulERAIDproject,in thatsomenew, youngscientistsweretrained and several successful patentshavecomeoutofthe project. Forexample, onedis coverywasavery strongenzymethatcan function in highalkalinity (pH 12)while degrading modified cellulose. This has already been incorporated in a Japanese detergentcalled"Attack." Anotherdiscovery hasexceededallconceptionsofwhat constitutes a viable environment, an unusual, feisty life-form that can thrive in a 50% solution ofvery toxic toluene, in sharp contrast to ordinary organisms which perishinasolutionofjust0.1%. WewereabletoisolatetheDNAfromthetoluene resistant bacterium and introduce it into toluene-sensitive mutants. It should ulti mately bepossibletoisolatethespecial genesand transfersolvent-tolerance. Inmorerecenttimes,manyorganismshavebeenfoundinextremeenvironments. Moreover, some ofthem cannot survive in so-called moderate environments. For example,thermophilicbacteriagrowinenvironmentswithextremelyhightempera tures,butwillnotgrowat20°Cto40°C. Somealkali-lovingbacteriacannotgrowin anutrientbrothat pH7.0,butflourish atpH 10.5. Barophilicmicroorganisms iso lated from the MarianaTrench absolutely require higherpressures (higher than 80 MPa) for growth. Ifa moderate environment for conventional organisms such as Escherichiacoliweresuperimposedonthatforextremophiles,the"moderate"envi ronment would be anextremeenvironment. Thus, the ideaofan extreme environ mentisrelative,notabsolute. Clearlywehavebeentooanthropocentricinourthink ing. Weshouldthereforeextendourconsiderationtootherenvironmentsinorderto isolateandcultivatenewmicroorganisms. A new l5-year research program called International DEEP STAR (Qeep-sea EnvironmentExplorationfrogram,ScienceandTechnologyforAdvancedResearch) was launchedinOctober 1990inYokosukathroughJAMSTEC. Ihavebeenplaced in chargeofthis program and wouldlike to expand thesources ofmicroorganisms for study from thesurfaceofthe globetothedeepsea. JAMSTEChastwosubma rines (Shinkni 2000and Shinkai 6500), two unmanned submersibles (Dolphin 3K and Kaiko), and can collectsamples from the deepest parts ofthe oceans. The re- Preface VII motely controlled Kaiko was used tocollect samples from the deepest pointofthe Mariana Trench as described above. When I accepted the DEEP STAR post, my classmate,fonnerPresidentofJAMSTECIsaoUchidatoldme: "JAMSTECcannot study fish, because they belongto the Ministry ofAgriculture and Fisheries, and it can'tstudythestructureoftheseaorports, sincethis is undertheMinistry ofCon struction." JAMSTEC decided to tum to microorganisms "because they belong to nobody." SoIhadgoodscientificandeconomicreasonsfordirectingamajorproject tostudymicrobiallifeatthebottomofthesea. Onepotentialgoal istocollectnew infonnationonthe originsoflife. The program atYokosuka turns out to be rather unusual in that5 to 20million dollars a year has been spent since 1991. At JAMSTEC there are 30 researchers workingondeep-searesearch,andfrom 1992to 1998another 45 researchers from tencountriescametoworkwithus. Engineersandscientistsfrom thisprojectspent twoyearsconstructingan incrediblefacility thatallowsthemtocollectmicroorgan ismsunderdeep-seahydrostaticpressureand togrowthemcontinuously inculture, transferring them constantly under high pressure.These scientists also havedevel oped the capability of working at very high temperatures, to better preserve and studydeep-seahydrothennal-ventbacteria. Thegroups in DEEPSTARarefocusing on thefollowing areas: 1. Organicsolvent-tolerantmicroorganisms. Ittooktwoyearstoisolateanorgan icsolvent-tolerantbacteriumin 1989. However,in 1991 ourgroupdevelopeda sophisticatedmethodtoisolateorganicsolvent-tolerantbacteriafrom deep-sea samples. Using thismethod, theisolation frequency ofsolvent-tolerantmicro organisms was about 100times higherthan that ofthe isolation previously re ported. Someofthese microorganisms can completely digest crudeoil in sea water. This finding has paved the way to removing oil spills. These organic solvent-tolerantmicroorganisms haveconsiderablepotential for application in bioreactorsassolvent-tolerantmicrobial catalysts. 2. Hyperthermophiles. Morethan50strainsofhyperthennophileshavebeen iso latedfromhydrothennalventsintheOkinawaarea. Oneofthesehyperthenno philes is Pyrococcus horikoshii, isolated in collaboration with The Center of MarineBiotechnologyoftheUniversityofMaryland.The wholechromosomal DNAsequenceofthisstrainwasdetenninedbytheNational InstituteofTech nology and Evaluation in 1998. Several enzymes in hyperthennophiles have been investigatedfor industrialapplications. 3. Psychrophiles,bacteriathatgrowatvery lowtemperatures. Thesebacteriaare agoodresource toisolatenovelenzymesthatareactiveat lowertemperatures. 4. Barophiles, bacteria that grow only under hydrostatic pressure. DEEP STAR researchershavesucceededinisolatingmanyobligatelybarophilicbacteriathat function only under high pressure and have isolated the genes responsible for barophily. Phylogeneticstudiesoftheisolatessuggestedthattheisolatedstrains belongtoanewsub-branchofthegammagroup. Itwasdiscoveredthatthelac promoter was activated under high pressure in the absence of inducers. AI- VIII Preface though the enzymes produced by these barophiles have not been studied, it is highlylikelythatitwillbepossibletoclonenovelgenesfrombarophilesandto expressthem inconventionalmicroorganisms for industrial applications. Fur thermore,thephysiologyofSaccharomycescerevisiaehasbeenextensivelystud ied underelevatedhydrostatic pressurestoestablish barophysiology. 5. WholegenomeanalysisofalkaliphilicBacillushaloduransC-125,abacterium producing alkaline protease, alkaline cellulase, and alkaline xylanase. Alka liphily andthe environmental adaptation mechanism will beclarified from the whole DNAsequencedeterminedby "shotgunsequencing." 6. Work is proceeding on preservation and culture collection, and research has begunon thegeneticsofthesemarine bacteria. DEEPSTARresearchershavesuccessfulIymovedtothesecondphase(1998-2005) with thefollowingresults. I. Bytheendof1998about250articleshadbeenpublishedinrespectedjournals. 2. InJanuary 1998, DEEPSTARorganizedthe International CongressonExtre mophiles atYokohama. About 350participants, including 150scientistsfrom abroad, discussedextremophiles. 3. ThesciencejournalExtremophileswaslaunchedin 1997bySpringer-Verlag. wasappointedthefirstChiefEditor. This book is written to commemorate oureight-year studies ofdeep-sea microor ganisms. Someoftheworkhasnotbeenpublishedinscientificjournalsforvarious reasons: somebecauseitisnotyetready forsubmission,someforpatentandother reasons. However, thesepersonalreportsshouldbeuseful in thestudyofdeep-sea microorganisms. All the individuals involved have worked very hard to develop deep-seamicrobiology,includingbarophysiology. Iamextremelygratefulfortheir cooperationinthisprojectduringthefirst phase. I would also like to pay special thanks to former Presidents I. Uchida, andW. IshizukaandPresidentT.HiranoofJAMSTEC,whokindlygavemetheopportunity toexpandthe new worldofextremophiles. K. Horikoshi Contents Preface V Part 1 Biodiversity in Deep-Sea Environments I Isolation and Characterization ofMicroorganisms from Deep-SeaMud HidetoTakami 3 2 MolecularAnalyses ofthe Sediment and Isolation of Extreme Barophilesfrom the DeepestMarianaTrench Chiaki Kato 27 3 Taxonomy and BiotransformationActivities ofDeep-Sea Actinomycetes AlanT. Bull, Joy A. Colquhoun, and Stephen C. Heald 39 4 Microbial Diversity in the Sediments Collected from Cold-SeepAreas and from Different Depths ofthe Deep-Sea LinaLi andChiaki Kato 55 IX X Contents Part 2 Adaptation and Response of Extremophiles Toward Extreme Deep-Sea Conditions 5 Barophiles (Piezophiles) Chiaki Kato 91 6 Thermophiles Juan M. Gonzalez 113 7 Deep-Sea Psychrophiles TetsuoHamamoto 155 8 Organic Solvent-Tolerant Microorganisms AkiraInoue................................................................................... 175 9 Microbial ProcessesAssociated with aMethane Seep Tubeworm Takeshi Naganuma 211 Part 3 NewApproaches and Future Scope for Deep Sea Microbiology 10 Barophysiology (Piezophysiology) FumiyoshiAbe andChiaki Kato 227 11 Genome Analysis ofFacultativelyAlkaliphilic Bacillus halodurans C-125 HidetoTakami 249 12 Future Scope Kaoru Tsujii 285 Appendix: International Congress on Extremophiles '98 at Yokohama> '" 293 Appendix: List ofPublications ofthe First DEEPSTAR Project: October 1, 1990 to September 30, 1998 301 Subject Index 311 Contributors Koki Horikoshi TheDEEPSTARGroup, Japan MarineScienceandTechnology Center, 2-15 Natsushima-cho,Yokosuka, Kanagawa 237-0061, Japan HidetoTakami TheDEEPSTARGroup, JapanMarineScienceandTechnologyCenter, 2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan ChiakiKato TheDEEPSTARGroup, Japan MarineScienceandTechnology Center, 2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan AlanT.Bull DepartmentofBiosciences,UniversityofKent, Canterbury, Kent,CT27NJ,UK LinaLi TheDEEPSTARGroup, JapanMarineScienceandTechnologyCenter, 2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan JuanM.Gonzalez CenterofMarineBiotechnology, University ofMarylandBiotechnology Institute, 701 E. PrattSt.,Baltimore,MD21202,USA TetsuoHamamoto BiotechnologyTransferServices, 4-14-9Kamiogi, Suginami-ku,Tokyo 167-0043,Japan AkiraInoue TheDEEPSTARGroup, Japan Marine ScienceandTechnology Center, 2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan XI
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