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2005 Volume 96 Marine Biotechnology I

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Adv Biochem Engin/Biotechnol (2005) 96: 1–48 DOI 10.1007/b135781

©Springer-Verlag Berlin Heidelberg 2005 Published online: 24 August 2005

Screening for New Metabolites

from Marine Microorganisms

Thomas Schweder (u) · Ulrike Lindequist · Michael Lalk

Institut für Marine Biotechnologie, W.-Rathenau-Str. 49, 17489 Greifswald, Germany Schweder@uni-greifswald.de

1 Introduction. . . 2

2 Sequencing of the Genomes of Marine Microorganisms. . . 3

2.1 Completed and Ongoing Marine Sequencing Projects . . . 3

2.2 Analysis of Marine Microbial Diversity . . . 18

2.3 Environmental Genomics . . . 18

2.4 Functional Genome Analysis . . . 22

2.4.1 Proteome Analysis . . . 22

2.4.2 Transcriptome Analysis . . . 23

2.5 Genome Sequencing and Identification of New Antimicrobial Compounds 24 3 Screening for New Metabolites. . . 26

3.1 Alternative Cultivation Methods . . . 26

3.2 Preparation of Materials for Screening . . . 27

3.3 Chemical and Physicochemical Screening . . . 28

3.4 Biological Screening . . . 28

3.5 High-Throughput Screening, Automation, Data Management . . . 30

3.6 Metabolome Analysis Techniques . . . 31

3.7 Examples for Metabolites from Marine Microorganisms . . . 32

4 Application of Proteomics for Target Analyses of Antibacterial Compounds 41 5 Influence of Cultivation Conditions on Metabolite Production . . . 41

6 Outlook . . . 42

References . . . 42 Abstract This article gives an overview of current analysis techniques for the screening and the activity analysis of metabolites from marine (micro)organisms. The sequencing of marine genomes and the techniques of functional genomics (including transcriptome, proteome, and metabolome analyses) open up new possibilities for the screening of new metabolites of biotechnological interest. Although the sequencing of microbial marine genomes has been somewhat limited to date, selected genome sequences of marine bac-teria and algae have already been published. This report summarizes the application of the techniques of functional genomics, such as transcriptome analysis in combina-tion with high-resolucombina-tion two-dimensional polyacrylamide gelelectrophoresis and mass spectrometry, for the screening for bioactive compounds of marine microorganisms. Furthermore, the target analysis of antimicrobial compounds by proteome or transcrip-tome analysis of bacterial model systems is described. Recent high-throughput screening

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techniques are explained. Finally, new approaches for the screening of metabolites from marine microorganisms are discussed.

Keywords Functional genomics· Proteome · Metabolome · Natural compounds ·

High-throughput screening

1

Introduction

The tremendous biochemical diversity of marine microorganisms and their biotechnological potential is becoming more and more recognized, not only by microbiologists but also by the pharmaceutical industry. Several new companies focus on the discovery of more effective drugs based on natu-ral products of marine microorganisms. In recent years, the improvement of screening technologies has yielded a considerable number of potential new drug candidates and other metabolites from microorganisms of marine ecosystems.

The physiological investigation of marine natural products has shown that many of these compounds function as signal transducers and thus regulate complex processes within marine living societies. It is supposed that these structures play an important role in the inhibition of physiological processes of potential competitors. This offers a promising potential for the exploration of new drugs against critical pathogenic microorganisms.

Most of the marine compounds that have been successfully screened and structurally elucidated so far originate from microorganisms, especially bac-teria. Several studies have demonstrated that natural products isolated from higher marine organisms like marine invertebrates are very frequently of bacterial origin. However, these bacteria, which are for example in a symbi-otic association with higher organisms, usually cannot be cultivated alone in a pure culture. Their growth depends directly on the activity of their hosts. Furthermore, only a minority of free-living marine microorganisms has been identified and can be cultivated so far. The knowledge on such marine mi-croorganisms is very limited compared to those mimi-croorganisms that can be easily cultured under laboratory conditions. The taxonomical identification of marine microorganisms in general is still in its infancy. The focus on the physiology and the potential of bioactive substances of non-cultivable marine microorganisms is an important challenge at present and for the future.

The estimated taxonomical diversity of marine microorganisms in general indicates the powerful potential of novel bioactive substances produced in aquatic ecosystems. It has been shown that marine bacteria, which are the predominant microorganisms in the marine ecosystems, produce bioactive substances that are different from known compounds from terrestrial

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bacte-Screening for New Metabolites from Marine Microorganisms 3 ria. Also, metabolites from facultative and obligate marine fungi often have structures unlike those of their terrestrial counterparts [1].

In the past, a successful cultivation was usually the prerequisite for the screening and final application of a new natural compound. However, during the last few years promising techniques have been developed that allow the screening and presumably also application of biological activities, not only of cultivable marine microorganisms but also of those organisms that cannot be cultivated at present. These techniques are the focus of this article.

2

Sequencing of the Genomes of Marine Microorganisms

The genome sequencing of an organism gives the blueprint of its life. This blueprint establishes the basis for a comprehensive view of the cellular phys-iology. Knowing the sequence of all genes does not only allow the identifica-tion of protein funcidentifica-tions but also makes it possible to explore the complexity of the cellular organization of an organism. Elucidation of the structural orga-nization of sequenced genomes has led to new insights into the physiological capacity of these organisms. This opens up new possibilities for the explo-ration of genes that are involved in pathways responsible for the synthesis of metabolites of biotechnological interest. However, the functions of the major-ity of genes are still unknown. Understanding these functions will be a major challenge for the next decades. In this field we are only at the beginning. 2.1

Completed and Ongoing Marine Sequencing Projects

A prerequisite for the sequencing of the whole genome of an organism is usually the cultivation of the cells in a pure culture. It is estimated that about 1–10% of the microbial diversity on earth has been identified [2]. However, the majority of the taxonomically classified microorganisms are of terrestrial origin. The considerably smaller number of identified marine microbial species is one of the reasons why most of the microbial genomes that have been sequenced so far originate from terrestrial microorganisms. Only few marine genome projects have been started and finished up to now. The first completely sequenced genomes of marine bacteria were that of the methanogenic bacterium Methanococcus jannaschii [3] and that of the cyanobacterium Synechocystis spp. [4]. A selection of completed and ongoing genome sequencing projects dealing with marine microorganisms is given in Table 1.

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Table 1 S el ec ti o n o f co m pl et ed an d o ng o ing m ar ine ge no m e se q u en ci ng p ro je ct s Co m p le te g eno me seq uenc es o f mar ine micr o o rg anisms O rga n ism P ro p er ti es Gen o me siz e (Mb ) In st itu tio n R ef er en ce Aq u if ex a eo li cu s V F 5 H y pe r-th er m o phi li c m ar in e 1. 551 D ive rs a, [5 ] ba ct er iu m (8 5 – 95 ◦C) Sa n D ie go /USA Ae ro p yr u m p er n ix A er o b ic h y p er th er mo p h ilic ar ch aeo n 1.6 69 N at io n al In st itu te o f [6 ] gr o w in g at te m p er at u re s u p to 100 ◦C Tech n ol og y an d E va lua ti o n (N ITE ), Japan Al ca n iv or a x b or ku m en si s O il-d eg rad in g (h yd ro car bo n o cl as ti c) , 3.1 2 C o m p et en ce N et wo rk [7 ] su rf ac ta n t-p ro d u ci n g ma ri n e B ielef eld , Ger m an y ba ct er iu m A n a b aena sp . st ra in P C C 7120 C ya n o b ac te ri u m 7. 212 Ka zu sa DN A R es ea rch [8] (1 ch ro mos o me o f 6.414 M b an d In st itu te , Japan si x p la smi d s o f 408.101 bp , 186.614 bp , 101.965 bp , 55.414 bp , 40.340 bp an d 5.584 bp ) Ca rb ox yd ot h er m u s Th er mo p h ilic an aer o b ic b ac ter iu m 2.1 TIGR , C en te r o f M ar in e [9 ] h yd ro geno fo rm a n s fr om h yd rot h er m al en v iron m en ts Bi ot ec h n o lo g y, U SA Ch lo ro bi u m te pi d u m T L S Ph o tos y n th et ic , an aer o b ic , 2.1 55 TIGR , U SA [1 0] g re en -s u lf u r b ac te ri u m , the rm o phi le

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Screening for New Metabolites from Marine Microorganisms 5 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce Col w elli a p sy ch ro er yt h rae a 34H A rct ic b act er iu m 5. 3 T IG R , U SA [1 1] M eth a n oc oc co ides bu rt on ii A n ta rc ti c archa ec ho n (o n e o f tw o 2. 8–3 Jo in t G eno m e In st it ute , [12] co ld -a d ap te d ar ch ae an s to b e U SA seq u enc ed) M eth a n oc oc cus ja n n a sc h ii M eth an o ge n ic ar ch eo n 1.6 6 T IG R, U SA [1 3] D SM 2661 E xt re m o phi le , g ro w s ne ar hy d ro th er m al ve n ts at p re ss u re s o f m o re th an 200 at m and op ti m u m te m p er at u re o f 85 ◦C M etha n op yr us ka nd leri AV 19 H y pe rt he rm o phi le m et h an e-1. 695 Fi de li ty Sy ste m s, Inc, [14] p ro d u ci n g archa eo n at the b ase USA o f b la ck smok ers (t em p era tur es ne ar 100 ◦C) N a n o arch a eum equit a n s Ma ri n e ar ch ae on , liv in g as an 0. 49 1 D iv er sa C or p o ra ti on , [1 5] ob li ga te sy mbi o n t on an ot h er Sa n D ie go /USA micr o b e (Ig ni co cc u s)i n u n d er se a ve n ts and ho t sp ri n g s (o ne o f th e sma ll est ge no mes o f an y seq u enc ed micr o b e) O ce a no b a cill us iheyen si s H TE831 Ba ci ll us-rela ted d eep sea sed imen t 3.6 Ja p an M ar in e Sc ien ce [1 6] b ac ter iu m, to lera tes ext remel y sa lin e an d Tec h n olog y C en ter an d al k alin e en v ir o n m en ts

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce P roc h lo roc oc cus m a ri n us SS1 20 M ar in e cy an o b act er ium , o n e o f th e 1.8 6 G en o sc o p e an d Sta ti o n [1 7] m o st ab u n da n t pho to sy n the ti c B io lo g iq u e d e R o sc o ff , o rgan is m s o n ear th F ran ce L o w-li g h t-ad ap te d P ro ch lor o co cc u s m a rin us M IT9313 L o w -l ig h t-ad ap te d ec o ty pe (M IT 9313) 2. 411 Jo in t G en o m e In st it u te , [18] in d eep er wa te rs w ith less su n li g h t USA P roc h lo roc oc cus m a ri n us su bs p. H ig h ly li g h t-ada p te d ec o ty pe 1. 658 Jo in t G en o m e In st it u te , [18] past or is C C M P1378 (M ED 4) (MED4 ), n ea r th e o cea n sur fa ce USA P yr obac u lu m a er op h il u m D C 3000 F ac u lt at iv el y aer o b ic n it ra te-2. 2 U n iv ersit y o f C al if o rn ia [1 9] re d u ci ng h y pe rt he rm o phi li c & C al ifo rn ia In st it ute cr en ar cha eo n , T (o p t) = 100 ◦C, o f Te ch n o log y, U SA is o la te d fr om a b oi li n g m ar in e wa te r h ole P yro co cc u s a b ys si G E 5 A rcha eo n , g ro w ing cl o se to ho t 1. 765 G eno sc o pe , F ra n k re ich [20] spr in g s 3500 m d eep in th e so u th eas t P ac ifi c (o p ti m al ly at 103 ◦C and 200 at m o sphe re s) P yr o co cc us fu ri o sus D SM 3638 A rc h ae o n fo un d in m ar in e san d 1.9 08 U n iv er si ty o f U tah [2 1] su rr o u n d in g sulf u ro us vo lc an o es , & U n iv ers it y o f o p ti m al g ro w th at te m pe ra tu re s M ar yl an d, U SA ab o ve 100 ◦C , hi g h ly re si st an t to ra di at io n

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Screening for New Metabolites from Marine Microorganisms 7 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce P yro co cc u s h or ik o sh ii O T 3 H y pe rt he rm o phi li c archa eb ac te ri u m 1. 739 N at io n al In st it ute o f [22] n ear h yd ro th er m al ve n ts , Te ch n o lo g y an d ext remo p h ile (o p tim um g ro w th E va lua ti o n ,J ap an te m p er at u re 98 ◦C) Pyr ol ob u s fu m a ri i A rc h ae o n , o p tim al g ro w th at 106 ◦C 1.8 D iv ers a, C elera [2 3] (in h yd ro th er ma l ven ts at th e b o tt o m G en o m ic s, USA o f th e A tlan ti c O ce an ) Rhod op ir ell ula b a lt ica M ar ine pl an ct o m yc et e fro m the 7. 145 M ax P la nck In st it u te [24] (f o rmerl y P ir ell ula sp . st ra in 1) B al ti c S ea , emits a red d ish su n sc reen o f M ar in e Mic ro b iolog y, to p ro tec t itself fr o m sun li g h t G er ma n y Su lfol ob u s tok o d a ii 7 A ero b ic the rm o aci do phi li c 2. 695 N at io n al In st it ute o f [25] cr en ar ch ae o n , o p tim al g ro w th Te ch n o lo g y an d E va lu at io n at 80 ◦C (h o t sp rin g s), at lo w p H (NITE), Ja p an Sy ne ch o co cc u s W H 8102 M ar ine u n ic el lu la r cya no b ac te riu m , 2. 4 Jo in t G eno m e In st it ute [26] pho to sy n the ti c, m o ti le , in the an d Scr ip ps In st it ute , o p en o cea n U SA Sy ne ch o cy st is P C C 6803 U n ic el lu la r aq u at ic , p h o to sy n th et ic 3.5 73 K az u sa D N A R esea rc h [2 7] cy an o b act er ium In st itu te , Japan Ther m o sy ne ch o co ccus T h er m o phi li c u ni ce ll u la r cy an o b ac te ri u m 2. 594 Ka zu sa DN A R es ea rch [28] elo n ga tus B P -1 (ho t sp ri ng s) In st it u te, Ja p an

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce T h er mo to ga ma ri ti ma MSB 8 E xt re m el y the rm o phi li c eub ac te ri u m 1. 861 T h e In st it u te fo r [29] gr o w in g u p to 90 ◦CG en o m ic R es ea rc h (T IG R ), Ma ry la n d/ USA V ib ri o sc h er i E S114 M ar ine b io lu m in es ce n t b ac te riu m , 4. 136 In te g rate d G eno m ics [30] sp ec ifi c sy m b io n t in th e li g h t-In c, U n iv . o f H aw aii, em it ti n g or ga n s of ce rt ai n sp ec ie s U SA o f sq u ids an d fi shes V ibrio vu ln ificus H al o phi li c se aw ate r p at ho ge n, 5. 261 (2 chro m o so m es o f Ya n g-Mi ng U n ive rsi ty , [31] ca us es wo un d in fect io n s, 3.355 Mb an d 1.857 Mb , o ne T ai w an ga st ro en ter it is p la smi d o f 48.508 bp ) Geno mes and chr o moso mes o f m ar ine m icr o o rg anisms in p ro g ress A erom on a s h yd rop h il a En te ro to xic aer o m o n ad , fi sh N o d at a T IGR , U n iv ersit y [3 2] b act er ial p at h o ge n o f M ar ylan d , U SA A er omo n a s sa lmo n icida Sa lmo n id b act er ial p at h o gen 4.7 N at io n al R es ear ch C o un ci l, [3 2] su b sp . sa lm on ic id a In st it ute for M ar ine Bi o sc ie n ce s, C ana da Bac ter io vor a x m a rin u s SJ M ar ine p re dato ry b ac te ri u m 3. 431 Sa ng er In st it ute , [32] (f o rmer sa lt w at er B d ello v ib rio sp .) th at p aras it is es a w id e ran ge U n iv ers it y o f M ar ylan d , o f G ram n ega ti ve b act er ia M o rgan St at e U n iv ers it y, USA

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Screening for New Metabolites from Marine Microorganisms 9 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce C h lor ob iu m limi co la D SM Z 245( T ) Gr een su lf ur b ac ter iu m 2.4 Jo in t G en o m e In st itu te , [3 2] USA C h lor ob iu m ph ae obac ter oi d es Gr een su lf ur b ac ter iu m 2.4 Jo in t G en o m e In st itu te , [3 2] D SM Z 266( T ) a n d M N 1 USA Ch lo ro bi u m v ibr io fo rm e Gr een su lf ur b ac ter iu m 2.4 Jo in t G en o m e In st itu te , [3 2] f. th io sul fa top h il u m D SM Z 265( T ) USA C h lo ro flexus a u ra n ti acus J-10-fl P h ot ot ro p h ic g li d in g fi la m en to u s 3. 0 Joi n t G en o m e In st it ut e, [3 2] ba ct er iu m o f h ot spr in g s U SA C ena rc hae u m sy m b io su m P sy chro phi li c cre na rcha eo n that 3. 7 M o n te re y B ay A q u ar iu m [32] in h ab its a m ar in e sp o n ge R es ear ch In st itu te , U SA Cr o co sp h aer a w at son ii W H 8501 D ia zo tro pi c m ar in e cya no b ac te riu m , 4. 0 Jo in t G eno m e In st it ute , [32] is ol at ed fr o m th e tr o p ica l A tl an ti c W o o d s H ol e O ce an og ra p h ic an d P ac ifi c O ce an s In st itu ti o n , U SA D ei n oc oc cus ge oth er ma li s E xt remel y ra d ia ti o n -r esista n t N o d at a Jo in t Gen o me In st itu te, [3 2] D SM 11300 an d sl ig h tl y the rm o phi li c U SA b ac te ri u mf ro mh o t sp ri n g

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce D esul fobac teri u m a u to tr op h ic u m O ri g in al ly is ola ted fr o m > 5. 5 G ö tt ing en G eno m ics [32] HR M2 ma ri n e m u d o f th e L ab o ra to ry , R ea l M ed it er ra n ea n S ea En v ir o n m en ta l G en o m ic s (REGX, M ax P la n ck In st itu te fo r M ar in e M ic ro b iolog y, B re men ), G er ma n y D esu lfot a le a psy ch rop h il a LS v5 4 P sy chro phi li c su lfate -re d u ci n g 3. 66 E pi da u ro s Bi o te chno lo g ie [32] b ac ter iu m is o la te d fr o m A G, REGX, G er ma n y pe rm an en tly co ld A rc ti c ma ri n e sed imen ts D esul fu rom onas ac et ox id a n s An ae ro b ic , su lf ur -r ed uc in g , 4. 1 Jo in t Gen o me In st itu te, [3 2] ac eta te-o xid iz in g b act er ium U SA fr o m sea o r fr esh w ater Edw a rd si ella ic ta lu ri 93-146 P rim ar y b act er ial pa th o ge n N o d at a O k lah o m a U n iv ers it y [3 2] o f ch an n el ca tfish H ea lt h Sc ien ce C en ter , Mi ssi ssi p p i St ate Un iv er si ty , U SA E pulo pi sci u m sp . L ive s in the in te st in es N o dat a T IG R [32] of se ve ra l k in d s of su rg eo n fi sh o ff the A u st ra li an sho re, up to 500 µ mi n si ze

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Screening for New Metabolites from Marine Microorganisms 11 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce F la vobac te ri um psy ch rop h il u m Fish p ath ogen (c old w at er N o d at a N at io n al C en te r fo r [3 2] d is eas e) in aq uac u lt ur e C o o l an d C o ld W at er A q uac u lt ur e, In te g ra ted Geno mics, U SA Ha hella ch ej uen si s 96C J10356 E xt rac el lu lar p o ly sac ch ar id e-7. 0 K o re a R es ear ch In st itu te [3 2] p ro d uc in g m ar in e b act er ium o f B io sc ie n ce an d B io tech n ol og y H a loa rcula m a ri sm or tu i E xt remel y h al o p h ilic ar ch aeo n 2.7 U n iv ersit y o f M ar yla n d [3 2] A T C C 43049 fr o m th e D ea d S ea B io tec h n olog y In st itu te , In st it u te fo r Sy stem s B iol og y, USA H yp er ther m us b u ty licus H y pe rt he rm o phi li c p ep ti de -1. 9 E p id au ro s B io te chno lo g ie [32] fe rm en ti n g su lf ur ar ch ae b act er iu m A G , G er m an y, fr om th e se a fl o or of U n iv er si ty of C o p en h ag en , so lf at ar ic h ab ita ts Den m ark H yp h om on as n ep tu n ium Pr im ar y co lon iz er s o f su rfa ce s in 2. 7 U n iv er si ty of G eor g ia , [3 2] A T C C 15444 m ar ine en v iro nm en ts an d in are as T IGR , U SA ad jac en t to h yd ro th er m al ve n ts Ma gn eto sp ir il lum Is o late d fr o m m icro ae ro b ic N o dat a M ax -P la nck -In st it ute [33] gr yp h is w a ld en se zo ne s o f fre sh w ate r se d im en ts o f M ar ine Mi cro b io lo g y, M ax-P la n ck -I n st it u te of G en et ic s, G er m an y

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce M a gn et o sp ir il lu m m a gn et ot a ct ic u m F ro m mic ro aer o b ic zo n es 4.5 Jo in ed Gen o me In st itu te, [3 2] M S-1, A T C C 31632 o f fr esh w at er sed imen ts U SA M eth a n oc oc cus Th er mo p h ilic n it ro gen fi xi n g N o d at a M olec ular Dy n amic s, [3 2] ther mo li tho tr op h icus ar ch ae o n fr o m su b m ar in e U K In te g ra te d h yd rot h er m al ve n ts G en om ic s, U SA M eth a n oc oc cus vo lta e A rc h ae o n fr o m su b m ar in e N o d at a M o le cular Dy n am ic s, [3 2] h yd ro th er m al ve nt s Int eg ra te d G en o m ic s Inc , U SA Me th a n o ge n iu m fr ig idu m P sy chro phi li c, H2 -u si ng 2–2. 5 U niv . o f N ew S. W al es , [12] m eth an o ge n ic ar ch ae o n fr o m A u st ralian G en o m e R es ear ch A ce L ak e, A n ta rc ti ca F ac il ity , A u str al ia M o lec u lar D y n amic s, U K M et h an o sarc in a a cet iv or an s A ce to tro phi c m et ha ne -p ro d u ci ng 4. 1 G ö tt ing en Ge no m ics [32] b ac te riu m is o la te d fro m L ab o rato ry , G er m an y ma ri n e sed imen ts M eth yl op ha ga tha lassi ca S1 M ar in e meth yl o tr o p h N o d at a In teg ra te d G en o m ic s In c, [3 2] (1995-b p USA Plas mid ) M icr ob ulb ifer d eg rad a n s 2-40 A ero b ic m ar in e b ac te ri u m 6. 0 Jo in t G eno m e In st it ute , [32] th at de g ra d es an d re cy cl es U SA co m p le x ca rb o h ydr ate s

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Screening for New Metabolites from Marine Microorganisms 13 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce M ic ro cy st is a er ug ino sa P C C 7806 B lo o m -fo rm in g to xi c 4. 8 In st it u te P aste u r, F ra n ce [32] cy an o b act er ium M ycoba ct er ium m a rinum M Cl o se re la ti ve o f M. tu b er culosi s, 6. 739 Sa ng er In st it ute , UK , [34] fish and h u m an p at ho ge n U nive rs it y o f W as hi ng to n, USA, In st it ute P as te u r, F ra nc e, M o na sh U n iv ., A u st ra li a, U n iv . o f Ten n essee, USA N a u tilia sp . A m-H A n aer o b ic , th er mo p h ilic sulf u r-N o d at a U n iv . o f Dela war e TIG R , [3 2] re d u ci n g b ac ter iu m, is ola ted fr o m USA tu b es o f th e d eep -s ea h yd ro th er m al ve n t po ly cha ete A lv ine ll a p o m pe ja na P ela g ibac ter u b iq ue H T C C 1062 A lpha pu rp le b ac te riu m , 1. 54 Ore go n St ate U niv ., [32] d o min an t micr o o rga n ism in th e D iv ersa , C en ter o f o ce an su rfa ce (b ac te ri o p la nk to n) M ar ine Bi o te chno lo g y, USA P ersep ho nella m a ri na Th er mo p h ilic h yd ro gen -o xid iz in g 1.6 P o rt lan d St at e U n iv ., [3 2] mic ro aer o p h ile fr o m d eep -s ea TIGR , U SA hy d ro th er m al ve n ts

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce Ps eu d o alt ero m on a s h al op la n kt is A n ta rc ti c p sy ch roto le ra n t T w o G en o sc o pe , F ra nc e [35] sea w at er b ac ter iu m ch ro m os o m es of ap p rox . 2700 an d 800 kb Re nib a ct er ium salm onin a rum A ct in o b ac ter iu m, ca us at iv e N o d at a N W F SC , N CCCW A, U n iv . [3 2] age n t o f b act er ial k id n ey o f W as h in g to n , In te g ra te d d is eas e in salm o n id fi sh es G en o m ic s, U SA R o se obac te r sp . TM 1040 Is o la te d fr om eg g s an d acce ss or y N o d at a Joi n t G en o m e In st it ut e, [3 2] ni da m en ta l g la n ds o f sq u ids USA Sh ew a nella ba lt ic a O S1155 Gra m n ega ti ve ga mma N o d at a Jo in t Gen o me In st it u te, [3 2] p ro teo b ac ter iu m fr o m th e USA Bal tic S ea Shew a nella fr ig id im a ri n a N C M B400 A n ta rc ti c G ra m n eg at ive N o dat a Jo in t G eno m e In st it ute , [32] ga mma p ro teo b ac ter iu m U SA Sh ew a nella v io la ce a DSS1 2 D ee p -se a b aro phi li c She w ane ll a 4. 9 K ink i U niv ., JAMS T EC , [32] st rain K eio U n iv ., Ja pan

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Screening for New Metabolites from Marine Microorganisms 15 Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce Silici b a cter p omer oy i DSS-3 D imeth yls u lf o n io p ro p io n at e-4.4 U n iv . o f G eo rg ia, [3 2] de m et h yl at in g b ac te ri a fro m T IG R , USA m ar ine en v iro nm en ts Sp h in gopy x is a las ke n si s RB2256 C h lo ro phe no l-de g ra d in g alpha N o dat a Jo in t G eno m e In st it ute , [32] P ro teo b ac ter iu m fr o m sea wa te r U SA fjo rd s in Alas k a Sp ir uli na pla ten si s Bl ue-g reen mic ro alga e fr o m N o d at a H uma n Gen o me C en ter , [3 2] la k es w it h h ig h sa lt B ei ji ng , C hi na con ce n tr at io ns , h ig h n u tri en t-d en si ty T h er m o co cc u s ko d a ka ra en si s K O D 1 E xt remel y th er m o p h ilic N o d at a K yo to U n iv . K wa n sei [3 2] he te ro tr o phi c archa eo n, G ak u in U n iv ., Ja p an o cc u rr in g in h ea te d sea flo ws Ther m ot o ga ne apo li ta n a E xt re m el y the rm o phi li c 1. 8 T IG R , U SA [32] A T C C 49045 eu b ac ter iu m n ea r d eep sea ven ts Th io m icr os pi ra cr u n ogena A u to tr o p h ic ga mma N o d at a Jo in t Gen o me In st it u te, [3 2] p ro teo b ac ter iu m fr o m U SA h ydro the rm al ve n t si te s

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Table 1 (c o n ti n u ed ) O rga n ism P ro p er ti es Siz e (Mb ) In st itu tio n R ef er en ce T ri cho d esmiu m er yt h ra eu m IM S101 M ar in e fila m en to us 6. 5 Jo in t Gen o me In st itu te, [3 2] cy an o b act er ium in tr o p ic al W o o d s H o le O ce an o g rap h ic an d su b tr o p ic al o ce an In st itu te , U SA U n cul ti va te d _ R ift ia p a ch yp lila _ end os ym bio n t Sy m b io n t o f d eep se a tu b e 3.6 – 4.0 M o lec u lar D y n amic s, [3 2] w o rm n ea r h yd ro th erm al Sc ri p p s In st it u ti o n ve n ts and ho t sp ri n g s o f Oc ea no g ra ph y, Qu o re x, U SA V ibrio sa lm on icida F is h p at h o ge n N o d at a U n iv . of T roms o , [3 2] NORS TR UCT , N o rw ay

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Screening for New Metabolites from Marine Microorganisms 17 Recently, the genomes of three different members of the cyanobacterium species Prochlorococcus marinus have been sequenced [36, 37]. These mem-bers of the genus Prochlorococcus dominate phytoplankton communities in most tropical and temperate open ocean ecosystems [38]. P. marinus is found in two main ecological forms: high-light-adapted genotypes in the upper part of the water column and low-light-adapted genotypes at the bottom of the illuminated layer [36]. Analysis of the genome sequences of the different P. marinus strains led to the identification of genes that play a role in deter-mining the relative fitness of the ecotypes in response to key environmental variables [37]. Furthermore, the genome comparison allowed determination of a putative minimal gene set of this photoautotrophic bacterium [36].

Glöckner et al. (2003) reported the complete genome sequence of Pirellula sp. strain 1 (“Rhodopirellula baltica”), a marine representative of the glob-ally distributed and environmentglob-ally important bacterial order Planctomyc-etales [39]. With 7.145 megabases, Pirellula sp. strain 1 has the largest circular bacterial genome sequenced so far. The genome sequencing indicated the presence of all genes required for heterolactic acid fermentation, key genes for the interconversion of C1 compounds, and 110 sulfatases, which were un-expected for this aerobic heterotrophic isolate.

The first genome of an uncultivable marine bacterium, the symbiont of the deep sea tube worm Riftia pachyptila [40] is currently being sequenced by a Californian consortium (Horst Felbeck, personal communication). This uncultivable endosymbiotic bacterium seems to form a monoculture in the trophosome of R. pachyptila. The knowledge of the bacterial genome se-quence together with molecular techniques such as the proteome analysis might help to find the explanation for this exclusive colonization by the mi-crobial endosymbiont inside the trophosome of the tube worm. Furthermore, the functional genome analysis of the bacterial symbiont will lead to the identification of mechanisms and possibly natural products involved in estab-lishing and maintaining the symbiosis with R. pachyptila.

Another promising field is the genome analysis of marine phages [41]. Phages have evolved unique strategies that sustainably influence the cellular processes of bacteria. The directed isolation of marine phages and the de-termination of their genome sequence could help to establish new strategies for the inhibition of growth of pathogenic marine bacteria, but also of hu-man pathogens. Such an approach has been undertaken by Liu et al. (2004) for Staphylococcus aureus phages [42]. The sequencing of 26 phage genomes led to the identification of 31 novel polypeptide families, the expression of which inhibited the growth of S. aureus. Furthermore, the analysis of poten-tial targets of these polypeptides led to the determination of proteins essenpoten-tial for bacterial DNA replication. A subsequent screening for small molecule in-hibitors of these targets allowed the isolation of chemical compounds that imitate the growth-inhibiting effect of selected phage polypeptides. Thus, this

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phage genomic approach could be a further strategy for the identification of new antibiotics to successfully combat infectious diseases.

2.2

Analysis of Marine Microbial Diversity

The majority of marine microbial organisms cannot be cultured under ar-tificial laboratory conditions and is thus not accessible for detailed taxo-nomical and physiological characterizations. However, advanced molecular techniques have altered the perspective on naturally occurring diversity and distribution of such marine microorganisms. Direct isolation of DNA from the environment makes it possible to identify bacterial species in different natural marine habitats without the cultivation of the microbial cells. This analysis is mainly based on the selective amplification of 16S rRNA gene se-quences by application of primers of conserved regions of the 16S rDNA in combination with the polymerase chain reaction (PCR) [43]. Due to its highly conserved and variable sequence regions the 16S rDNA sequence is used as a phylogenetic marker. The 16S rDNA sequence allows the identification and taxonomical affiliation of different microbial strains from an environmental sample. This analysis is thus an important feature, which allows the iden-tification of so far unknown organisms and gives valuable information on the biodiversity in marine habitats [44]. However, this technique gives only limited information on the distribution and physiology of the microorgan-isms in their natural environment.

The fluorescence in situ hybridization (FISH) allows investigations on the structure of a population without PCR amplification of specific sequences or cultivation of the microorganisms [45]. FISH with rRNA specific probes is a technique that allows phylogenetic identification of bacteria in mixed assemblages. For these analyses epifluorescence or confocal laser scanning microscopy are applied [47, 48]. The sensitivity of the FISH 16S rRNA analy-sis technique is based on the high number of ribosomes per cell and that each ribosome contains one copy of the 16S rRNA. This is a kind of natural signal amplification system.

2.3

Environmental Genomics

The FISH technique leads to a better understanding of the identity of selected microorganisms and their distribution and abundance in the appropriate ma-rine habitats. However, information concerning the biological properties of these marine bacteria cannot be satisfyingly illuminated this way. In this respect a new strategy has been developed, which is called environmental genomics. Similar to the sequencing of genomes from cultivable microor-ganisms, chromosomal DNA is used to generate genomic libraries. By the

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Screening for New Metabolites from Marine Microorganisms 19 environmental genomic strategy not only one genome is considered but all genomic sequences from one environmental sample. Large genomic DNA fragments are directly isolated from the environment and cloned into suit-able host vector systems. Establishment of comprehensive gene libraries at-tempts to cover all genome sequences from an environmental sample, to gather as much information as possible on the biosynthetic machinery of a microflora [49]. The comprehensive coverage of the genomes from an envi-ronmental sample is also called metagenome analysis. This technique allows a more realistic understanding of prokaryotic biodiversity in a distinct ma-rine habitat [50].

A prerequisite for establishing comprehensive genomic libraries from environmental samples is the availability of host vector systems allowing the stable propagation of large DNA fragments. For the cloning of large genome fragments usually fosmid [51] or bacterial artificial chromosome (BAC) [52, 53] vectors are used (Fig. 1). Fosmid or BAC vectors facilitate the conservation of large genomic fragments and thus open up the possibility to characterize not only the gene content but also the physiological potential of uncultivated microorganisms. The BAC system is based on the Escherichia coli single copy F factor plasmid. The E. coli F factor replicon allows for a copy number control of the clones so that they are maintained at one to two copies per cell. BAC vectors can carry DNA inserts of variable size with a maximum of 300 kb. Similar to the BAC system, the fosmid vectors also carry the E. coli

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F factor replicon, which provides for their stable single copy number. Fosmid vectors allow a size selection of the cloned DNA fragments from 32 to 43 kb by packaging the DNA inλ-phage heads. Shizuya et al. (1992) developed the bac-terial cloning system BAC for mapping and analysis of complex genomes [54]. Because of its high cloning efficiency and the stable maintenance of inserted DNA, the BAC system is able to facilitate the construction of DNA libraries of complex environmental genomic samples but also provides a comprehensive representation of the genome sequence of one organism. The stabilizing effect of BAC and also fosmid vectors is an important feature for the generation of comprehensive genome libraries since distinct regions of genomic DNA (e.g., coding for potential toxic proteins) can cause vector instabilities in high copy numbers.

The ability to clone long stretches of DNA has become an important tool for genome analyses of uncultivated marine microorganisms (Fig. 1). Stein et al. [51] isolated large genomic fragments from a widely distributed and rela-tively abundant, but as yet uncultivated, group of prokaryotes, the planktonic marine archaea from marine picoplankton. By construction and analysis of a fosmid DNA library a 38.5-kbp recombinant clone could be identified, which contained an archaeal small subunit ribosomal DNA gene. A similar approach was used to identify genomic DNA fragments of the symbiotic ma-rine archaeon Cenarchaeum symbiosum from the total DNA of the mama-rine sponge Axinella mexicana [55].

An environmental genomic study of Beja et al. [56] led to the discov-ery of proteorhodopsin, a retinal-containing integral membrane protein that functions as a light-driven proton pump, in the genome of an uncultivated marine bacterium. This study indicated that photoactive proteorhodopsin is present in oceanic surface waters. The data of Beja et al. (2001) suggested that proteorhodopsin-based phototrophy is a globally significant oceanic micro-bial process.

In another study Beja et al. [57] analyzed large genome fragments from microorganisms sampled from an Antarctic picoplankton population and compared them to those from deeper waters of the temperate North Pa-cific. This study was initiated to better characterize uncultivated planktonic crenarchaeotes, which are present in high abundance in Antarctic winter sur-face waters and deep ocean waters. For this purpose environmental genomic fragments were cloned into fosmid vectors and the inserts were sequenced. Analysis of the DNA insert of one Antarctic marine archaeon revealed dif-ferences in genome structure and content between archaea from Antarctic surface water and temperate deepwater. Analysis of the proteins encoded by the archaeon sequence from surface water and those derived from a deepwa-ter planktonic crenarchaeote revealed many typical archaeal proteins but also several proteins that have not been detected in archaea so far. Furthermore, a comparison of closely related archaea originating from a single population revealed significant genomic sequence differences that were not evident from

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Screening for New Metabolites from Marine Microorganisms 21 the 16S rRNA sequence analysis. Beja et al. [57] concluded that considerable functional diversity may exist within single populations of coexisting micro-bial strains, even those with identical 16S rRNA sequences.

Metabolic features are often coded in gene clusters, so called genomic is-lands. Therefore, the metagenome approach can also be applied to identify new genetic pathways for the directed synthesis of metabolites or enzymatic functions. Such functional genomic islands from uncultured microorganisms can be screened by isolating genetic material directly from original environ-mental samples (Fig. 1). The successful cloning and transformation of these sequences into a suitable host vector system for its expression and final char-acterization is a prerequisite for such screening approaches. One example in this respect is the commercialization of polyunsaturated fatty acids (PUFAs) from marine microorganisms. The most important PUFAs are eicosopen-taenic acid (EPA) and docosahexaenic acid (DHA). PUFAs are of biotechno-logical interest because of their beneficial properties to human health and their importance in infant development [58]. The screening of microbial PUFA synthesis genes from metagenome libraries allows for the cloning of the responsible genes into suitable expression hosts. The cloning of a 38 kb gene cluster from Shewanella putrefaciens into E. coli and Synechococcus spp. resulted in a successful EPA production by these microorganisms [59, 60]. The potential knowledge of PUFA-related genes offers the possibility for con-struction of recombinant microbial cell factories suitable for an alternative production of EPA and DHA.

The metagenome approach can also be used to automate the screening of genes from nature, either to look for new technical enzymes or for other specified activities. The screening of specific enzymatic activities requires the cloning and expression of a single gene. However, the production of dis-tinct compounds, such as metabolites or antibiotics, is coded by a set of genes. The cloning of complex environmental DNA samples into a suitable host in combination with HTS methods is a good strategy for the screening and isolation of pathways for metabolic functions or the discovery of new bioactive natural compounds directly from the marine environment. One pi-oneer in this field is the company DIVERSA (San Diego, California). This company estimates that its gene expression (metagenome) libraries currently contain the complete genomes of over one million different microorgan-isms (http://www.diversa.com/). DIVERSA has developed a set of automated ultra HTS and enrichment strategies. The company uses two different screen-ing strategies to discover novel biomolecules: (1) expression-based screenscreen-ing for biological activity and (2) sequence-based screening by identification of specific DNA sequences of interest. Recently, DIVERSA obtained the patent rights on a strategy claiming the construction and screening of expression libraries from nucleic acid directly isolated from the environment utilizing a fluorescence activated cell sorter (http://www.diversa.com/). This approach is based on a robotic screening system, which uses high-density microtiter

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plates, capable of screening and characterizing of about one million clones per day. If a clone expresses an activity or contains a DNA sequence of inter-est, it is isolated for further analysis.

2.4

Functional Genome Analysis

The function of the majority of genes within the sequenced marine genomes is not well understood. Furthermore, even if the complete set of genes of a microbial cell is available, it is mostly not known how these genes are reg-ulated or how the proteins interact to express their functions. In order to assign potential functions to the genes of a genome, functional genome an-alysis techniques are used. These techniques include the expression profiling of the whole set of genes by using genomic DNA arrays and/or proteomics. These techniques are not only suitable for exploration of the functions of the proteins but also help to find new potential drug targets. Proteome and tran-scriptome analysis techniques have led to a shift from direct antimicrobial screening programs toward rational target-based strategies. Furthermore, these techniques allow the identification of essential genes for the synthesis of selected metabolites.

2.4.1

Proteome Analysis

The proteome technique is mainly based on two-dimensional protein gel elec-trophoresis (2D-PAGE), used for protein separation, and MALDI-TOF mass spectrometry, applied for protein identification. Mandatory for the proteome analysis by mass spectrometry is the availability of the complete genome sequence of the organism of interest. The proteome of only few marine mi-croorganisms has been investigated so far. Most of these proteome studies explore how marine bacteria adapt to alterations in their environmental con-ditions. One of the first proteome analyses was performed using a marine Vibrio [61, 62]. In these studies the response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphate and to simultaneous de-pletion of all these nutrients (multiple-nutrient starvation) was examined. Gross et al. (1994) investigated changes in the two-dimensional protein pat-tern of selected marine bacteria and fungi in response to high-pressure stress [63].

The determination of proteome signatures related to defined variations in the environmental conditions also allows the affiliation of unknown gene/protein functions into functional groups. Rabus et al. (2002) studied the proteome of the planctomycete Rhodopirellula baltica during growth with N-acetylglucosamine and glucose [64]. Analysis of the two-dimensional protein

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Screening for New Metabolites from Marine Microorganisms 23 patterns revealed the presence of several protein spots, which were only de-tectable in soluble protein extracts of cells grown with N-acetylglucosamine.

Lemus and Ngai (2000) examined alterations in the proteome of the Eu-prymna scolopes light organ in response to symbiotic Vibrio fischeri. 2D-PAGE identified changes in the soluble proteome of the symbiotic light organ in-duced by a specific response to the interaction with V. fischeri [65].

Lopez et al. (2002) suggested the application of proteomics for the iden-tification of marine species by the analysis of species-specific peptides from randomly selected dominant protein spots [66].

2.4.2

Transcriptome Analysis

Proteome analysis is frequently accomplished by expression profiling with DNA arrays. However, genomic DNA arrays have so far been designed for only a few marine microorganisms. One of the first expression profiling studies with marine organisms using a genomic DNA array was done by Okamoto et al. (2003), who performed a genome-wide analysis of redox-regulated genes in a dinoflagellate [67]. In this study, the effects of sodium nitrite (a reactive nitrogen species generator) and paraquat (a producer of re-active oxygen species) on gene expression in the marine dinoflagellate species Pyrocystis lunula were investigated using genomic DNA microarrays.

Beside the application of genomic DNA microarrays for gene expression profiling, the use of DNA chips, with probes for a selected number of gene sequences, for ecological surveys and the exploration of marine biodiversity has been suggested [68]. Wu et al. (2001) developed and evaluated functional DNA arrays with genes involved in nitrogen cycling from pure cultures and those cloned from marine sediments [70]. The authors could demonstrate that their model DNA array in principle reveals functional gene composition in natural microbial communities. However, the study also showed that more work is needed to improve the sensitivity and specificity of this method. The advantage of DNA chips in comparison to the FISH method would be that multiple numbers of probes can be applied in one hybridization experiment. This advantage of DNA chips is of special interest for the analysis of micro-bial communities with a complex biodiversity. Furthermore, in comparison to FISH this approach allows the application of multiple probes for a better con-trol of false-positive and false-negative results. However, because of the large unknown amount of genetic sequences in an environmental sample, DNA chips are not yet widespread in marine microbial ecological applications [68]. For a successful application of DNA chips in this field, the discrimination of single mismatches is crucial [69].

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2.5

Genome Sequencing and Identification of New Antimicrobial Compounds

The sequencing of the genomes of microbial human pathogens has fun-damentally changed the capabilities for antimicrobial drug screening [71] (Fig. 2). Most antimicrobial drugs used today are derivatives of structures originating from terrestrial microorganisms. However, these drugs have been applied for many years and a considerable number of pathogenic microor-ganisms have adapted to these structures by developing antibiotic resis-tances [72, 73]. Therefore, new lead structures allowing new mechanisms of

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Screening for New Metabolites from Marine Microorganisms 25 action, which are different from the established bioactive compounds from terrestrial microorganisms, are required.

Most of the antimicrobial compounds currently on the market were screened based on whole cell antimicrobial screening programs. By ap-plication of new genome-driven techniques more directed, target-based approaches are possible [71]. These new screening strategies are directly coupled to potential drug targets, which have been identified by genome se-quencing projects. Such antimicrobial targets are for example proteins that are essential for microbial growth or cell survival.

Another important drug target are proteins that are involved in the pathogenicity of the pathogenic organism. In this respect, comparative ge-nomics of pathogenic and non-pathogenic strains is a suitable approach for the identification of pathogenicity related proteins. For example, comparison of the genomes of uropathogenic E. coli strains with those of non-pathogenic E. coli and from other E. coli pathotypes revealed the existence of so-called pathogenicity islands [74–76]. A similar approach has been used to investi-gate the pathogenicity of the marine bacterium Vibrio vulnificus [77]. This halophilic marine bacterium is an etiologic agent of human mortality from seafood-borne infections. Genome sequencing and comparative analysis led to the identification of selected genomic regions that are typical for V. vul-nificus and the human pathogen V. cholerae. The genomic information of this pathogenic bacterium can not only be applied for a monitoring of Vibrio in-fections but will eventually also lead to the identification of virulence factors of V. vulnificus.

The sequencing of the genome of a microorganism that has been identi-fied as a potent producer of bioactive compounds allows the identification of the gene clusters involved in the pathways for the production of these natural compounds. Myxobacteria like Myxococcus xanthus and Sorangium cellulo-sum have increasingly gained attention as producers of natural products with biological activity [78]. However, these myxobacteria are difficult to handle in large bioreactors for the biotechnological production of the metabolites of in-terests. Therefore, Müller and co-workers from the University of Saarbrücken are trying to identify biosynthetic gene clusters of these gram-negative bac-teria by genome sequencing. In order to establish a biotechnological pro-duction of selected bioactive structures a suggestion is to clone the essential biosynthetic gene clusters of these myxobacteria into suitable hosts such as Pseudomonas or Streptomyces, which fit the demands of the myxobacterial genetics and biochemistry [78].

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3

Screening for New Metabolites

3.1

Alternative Cultivation Methods

Less than 5% of the viable bacterial cells in marine samples ultimately grow under standard culture conditions [79, 80]. Typically, nutrient concentrations and cell numbers in the marine environment are three orders of magnitude lower than in common laboratory media [81]. Based on this fact, Button et al. (1993) developed a new approach for the isolation of marine bacte-ria [82]. This group simulated the normal substrate concentrations but also the cell density limited environmental conditions and was able to isolate typical marine bacteria. Rappe et al. [81] have improved the technique of But-ton et al. [82] and applied microarrays from the cell cultures coupled with FISH. A high throughput procedure allowing the cultivation of the abundant group of the ubiquitous SAR11 marine bacterioplankton clade was used. The combination of the HTS and molecular analysis techniques was helpful in increasing the rate at which cultures could be obtained and identified [81]. Fresh Oregon coast seawater samples were diluted so that each well of a mi-crotiter plate was inoculated on average with about 22 microbial cells. The medium in the microtiter wells was sterile Oregon coast water supplemented with phosphate, ammonium, and defined mixtures of organic carbon com-pounds. This approach allowed isolation of 18 bacterial cultures, which were not cultivable under standard cultivation conditions and which had resisted any cultivation efforts in the past. Rappe et al. [81] thus confirmed the suit-ability of diluting natural microbial communities in very low nutrient media for the isolation of new marine microorganisms. Consequently, high similar-ity to the original environmental conditions of the samples is helpful for the isolation of novel microbial species. Low nutrient conditions and the estab-lishment of the selection of single cells for cultivation were also successfully applied for the isolation of novel halophiles from Red Sea brine [83].

Furthermore, it has been reported that a combination of FISH and mi-croautoradiography supports the determination of physiological activities of microorganisms without cultivation [84]. In this approach a radioactively la-beled substrate was used for the incubation of a complex microbial sample. Cells metabolizing the radioactive-labeled substrate were finally detected by FISH. By this technique the strains are not only taxonomically affiliated but it is also possible to gain information concerning their physiological activity and the utilization of distinct substrates.

There is a challenge of developing further new culture techniques that incorporate an understanding of the special environmental conditions of ma-rine organisms in their natural habitats. This includes variations of dissolved

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Screening for New Metabolites from Marine Microorganisms 27 organic matter, trace elements, or surfaces. In most cases essential interac-tions with other organisms of the marine biotope have to be considered. The growth and development of selected microorganisms could directly or indi-rectly depend on the association with other bacteria or algae in the marine environment. Therefore, the consideration of microbial consortia rather than purified cultures could be a further suitable strategy for screening for new microbial activities. Furthermore, most cultivation attempts are performed either in liquid media or on agar plates as batch cultures. Special cultivation conditions, such as permeable solid substrates that imitate sediments or con-tinuous cultivations in flow reactors, are rarely applied. Another interesting aspect is the potential addition of growth-supporting signal molecules. Bruns et al. (2002) used signaling factors like the autoinducer acyl homoserine lac-tone or cyclic AMP to support the culturing of marine microorganisms [85].

It is thought that microorganisms only synthesize their bioactive com-pounds under distinct environmental conditions. Thus, beside the cultiva-tion of these microorganisms the identificacultiva-tion of the most suitable growth conditions for a maximal production of secondary metabolites also has to be considered. The influence of culture conditions on secondary metabolite production has for example been demonstrated for cyclic and linear lipopep-tides of Cyanobacteria [86]. The competition of different microorganisms for limited natural resources is also supposed to support the synthesis of an-timicrobial compounds. In the marine bacterium Streptomyces tenjimariensis production of the antibiotic istamycin is induced by co-culturing with other marine bacteria [87]. Such antibiotics frequently inhibit the growth of com-petitors and thus play an ecological role in the suppression of competitive microorganisms.

3.2

Preparation of Materials for Screening

The screening results depend on the quality of screening material, collec-tion and storage of organisms, cultivacollec-tion, extraccollec-tion, storage of extracts, and preparation of test samples. A directed (preselected) screening offers bet-ter chances of finding inbet-teresting metabolites than an undirected (“blind”) screening. Such a directed screening could be based on ecological observa-tions (peculiarities like interacobserva-tions, special environmental condiobserva-tions), on traditional experiences (use in ethnomedicine), or search in novel organ-isms. Mode and solvent of extraction determine which substances are ex-tracted [88, 89]. Solid phase extraction is a suitable method for automated sample preparation [90].

Extracts make special demands on sample preparation and screening sys-tems because they are usually complex mixtures of compounds. The concen-tration of active compounds in crude extracts is often very low (1 : 1000). One extract could contain compounds with opposite effects or compounds with

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low effect, which are active only in combination. False-positive results could be found. Further problems could be the lack of solubility in physiological solvents, the coloring of an extract (difficulties in spectroscopic measure-ments), or the non-tolerability of crude extracts to some bioassays.

3.3

Chemical and Physicochemical Screening

Chemical and physicochemical screening is the search for new chemical structures regardless of their biological activities. The first step is the sepa-ration of compounds from a complex extract by chromatographic methods. In a second step, the chemical reactivity or physicochemical properties of the separated compounds are analyzed by spectroscopic methods (UV/VIS, MS, NMR) or by detection with special detection reagents in the TLC [91]. The development of HPLC-DAD-MS systems allows the specific detection of single components in a complex mixture (e.g., an extract), regardless of the background of other metabolites. Comparison with data bases allows the identification of known structures (dereplication). Finding of unknown peaks in a HPLC or TLC chromatogram stimulates the isolation of the com-pound giving this peak by chromatographic methods and allows structure elucidation of the isolated substance by spectroscopic methods like nuclear magnetic resonance spectroscopy (NMR) or mass spectroscopy (MS). Taking into account the growth of (marine) microorganisms in microtiter plates, the screening for secondary metabolites could also be done on this miniaturized scale [92].

The new metabolites found by chemical screening still have to be tested for their biological activities. Combination of HPLC-separation, bioassays, and on-line spectroscopy miniaturizes and accelerates the identification of bioac-tive metabolites in a complex matrix [93].

3.4

Biological Screening

During biological screening test samples (extracts, fractions, pure com-pounds, and compound libraries) are screened for their bioactivities in vitro and/or in vivo. In the case of extracts, active metabolites could be isolated by bioactivity-guided isolation processes. The finding of structurally known compounds (dereplication) in active extracts is possible. Biological screening methods have to be of relevance for the objective (e.g., for the therapeutic aim), highly sensitive, selective (insensitive to inactive compounds and to ubiquitous compounds), and reproducible. They should be characterized by high information content and reasonable costs [94].

In vitro tests could be done on a molecular or on a cellular level. The information content will increase from molecular to cellular level, but the

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Screening for New Metabolites from Marine Microorganisms 29 throughput is higher in molecular test systems (question of quantity vs. qual-ity). Assays that require careful interpretation but provide a lot of information per assay are ideal for marine natural products research [88].

Tests on the molecular level are based, e.g., on receptor systems (identifi-cation of those compounds which bind to one receptor) or on enzyme sys-tems (enzyme-catalyzed turnover rates). Tests on the genome, transcriptome, or proteome level will become more and more important. Targets of high pharmacological relevance are G-protein coupled receptors, tyrosine kinase receptors, nuclear hormone receptors, ion channels, proteases, kinases, phos-phatases, and transporter molecules. The detection of a reaction on the mo-lecular level could be done by biochemical assays (e.g., spectrophotometric measurement of the product of an enzymatic reaction), ligand binding assays (readout by labeling with a tracer) or functional assays (reporter gene assays quantifying the expression level of a specific reporter gene product, second messenger assays, two hybrid assays for measuring protein–protein interac-tions). Fluorescence-based assay technologies, isotopic labeling, colorimetry, and chemoluminescence are very often used as detection methods [95–97].

Cell-based assays are more complex and more physiologically relevant than tests on the molecular level. On the other hand they are still labor inten-sive and more difficult to validate than molecular assays [98]. Permanent cell lines have many advantages in handling compared to primary cell cultures. Examples for cellular assays are: (1) vitality and proliferation studies with tu-mor cells or other permanent cell lines for the detection of antitutu-mor effects or undesired cytotoxic effects, and (2) growth studies with bacterial or fun-gal cells (diffusion assay or dilution assay) for detection of antibiotic effects or morphological and biochemical investigations of special cell types, e.g., en-dothelial cells, muscle cells, or keratinocytes. The detection could be done by microscopic investigation, counting of cell number, measurement of electro-physiological properties, determination of metabolic capacities or membrane integrity (dye uptake assay), uptake of precursors into DNA (proliferation studies), or several biochemical parameters (protein content and so on).

Most marine samples have only been tested in a limited number of as-says. The true drug potential of many of these compounds may therefore not yet be fully realized. Papers about new chemical structures of marine com-pounds give only initial biotesting data. Their significance is often difficult to determine. Papers with systematic screening results obtained from extracts are rare. The goal of highlighting compounds that are likely to become clin-ical candidates is also complicated by the fact that pharmaceutclin-ical companies are naturally reluctant to talk about compounds in the early stages of devel-opment [88]. The results of detailed pharmacological and biochemical studies could therefore be found only for selected compounds.

A predominant portion of bioactive marine metabolites (from marine bac-teria and fungi, but also from invertebrates) is occupied by antitumor /cyto-toxic compounds. Other activities are antibacterial, antiviral,

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anti-inflamma-tory, and enzyme-inhibiting [99]. Some toxic metabolites could be useful as molecular tools.

3.5

High-Throughput Screening, Automation, Data Management

The use of high-throughput screening (HTS) methods can improve the ef-ficiency of biological tests [95]. HTS is characterized by very high numbers of test samples (more than 100 000 per day in UHTS), automated processes, single-time-point measurement instead of kinetic measurements, develop-ment of miniaturized and paralleled techniques (96-, 384- or 1536-well mi-crotiter plates), and more sensitive detection technologies with fast read time [96, 100]. Presently HTS or UHTS are designed to handle large libraries of pure compounds, e.g., obtained by combinatorial chemistry [101]. They mainly use isolated targets.

The development of new HTS methods for cell-based screening is in progress. Because of their higher complexity they could simultaneously de-liver information on multiple parameters, provide higher quality data, and give better information about function of bioactive principles. Reporter sys-tems are introduced into the cells, which are based on the expression of genes encoding proteins such asβ-galactosidase, luciferase, or alkaline phos-phatase. An alternative to the use of expensive mammalian cells is the expres-sion of a selected physiological process in microorganisms such as Saccha-romyces cerevisiae. Novel methods of single molecule detection, e.g., fluores-cence correlation spectroscopy (FCS) will improve the processes [95, 102].

High-content screening (HCS) becomes more and more important. This means that a smaller number of high-qualitative compounds (higher purity) is screened with higher performance (high information content).

HTS is performed mainly by the industry and seldom by academic groups. It focuses on the active principle in a distinct bioassay. The screens are usually run only for a limited length of time. Due to the above mentioned problems with extracts, HTS is not suitable for these samples and crude extracts derived from natural sources therefore only play a minor role in HTS [95]. A bioassay-guided fractionation of an extract could need longer time than the screening of the pure chemical library [88].

A way to overcome the problems of biogenic samples in HTS is the syn-ergistic use of natural product chemistry and combinatorial chemistry. By bringing together the structural value and complex molecular shape of nat-ural products isolated from a natnat-ural source with rational synthetic strate-gies of combinatorial chemistry and biochemistry, the success of screening processes (the chance to find new lead structures) could be significantly improved [103]. Thus one could randomly isolate pure compounds on the ba-sis of interesting chemical structures, produce substance libraries and then screen these libraries [88].

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Screening for New Metabolites from Marine Microorganisms 31 In every case, the results of HTS represent only the first step in a series of experiments; valid cell culture experiments and animal assays are necessary. ADMET properties (adsorption, distribution, metabolism, excretion, and to-xicology of a bioactive metabolite) have to be considered. Only compounds with positive results in primary HTS and in secondary assays could be de-clared as a “hit”. The hits have also to be structurally defined.

Derivatization of hits by chemical or biochemical methods could produce a “lead”, a series of hits for which the structure-activity relationship is shown and activity demonstrated in vitro and in vivo.

A new development in the field of HTS is represented by the High Throughput Pharmacological System™(HTPS), which was patented by Ax-iom Biotechnologies (San Diego, CA) [104]. This is a fluidics- based platform that uses viable cells and test compounds to identify active compounds. This approach allows for a very fast estimation of the potency of the com-pounds and a determination of their specificity. HTPS can be understood as an instrument for automated programming of complex pharmacological cell treatment protocols. Edwards et al. (2001) [105] have coupled HTPS to a flow cytometer using a plug flow coupling valve technology. Flow cytome-try is performed with fluorescent probes and allows an optical measurement of physiological parameters of individual cells or cellular macromolecules at a high rate [106]. HTPS flow cytometry facilitates a high-throughput multi-factorial screening of large compound libraries to increase the efficiency with which novel bioresponse modifying drugs, such as from marine microor-ganisms, may be identified and characterized [105]. In this approach a large compound library in microtiter plates is sequentially combined with cells and finally delivered to a flow cytometer for multiparametric analysis.

3.6

Metabolome Analysis Techniques

The metabolome is the final product of proteome activity including the total assembly of low molecular weight molecules in a cell. Its composition is deter-mined not only by the genetic information encoded in the genes, but also by a particular physiological and developmental state as well as by environmen-tal factors. The main technologies for metabolome analysis are MS and NMR spectrometry, often combined with chromatographic methods. These require minute amounts of sample and will accommodate individual components of highly varying chemical structures and physical properties.

Metabolome analysis is expected to become a valuable tool in the search for new metabolites from marine microorganisms and in investigation of their biological effects on the metabolome of other cells.

Two of the methods of choice for the evaluation of metabolic constituents of cells are the hyphenated techniques HPLC-NMR and HPLC-MS. These methods can be used for the identification of several individual

References

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