・本資料は2008年10月8日に開催した「科学技術と産業」国際シンポジウム2008（主催：経済産業省、ジェトロ）にて、Rita R.Colwell氏が講演された際のプレゼンテーション資料です。

This is Dr. Rita R.Colwell’s presentation material used in METI-JETR symposium “Boosting Science and Technology through Industrial Collaboration 2008”, which was held on October 8th,2008.

・ Colwell氏のご好意により、ジェトロが同氏から許諾を得てここに掲載しています。

JETRO posts it on the website with permission by and courtesy of Dr.Colwell.

・無断転載・複製を禁じます。JETRO reserve all rights and users may not otherwise reproduce, sell, publish,

distribute, modify, display, or use all or part of the material without the prior written permission of JETRO.

Source: DOE Joint Genome Institute © 2003

Employing Marine Biotechnologyto Preserve and Utilize

the Environment WiselyJETRO Symposium

Boosting Science and Technologythrough Collaboration 2008

Tokyo, JapanOctober 8, 2008

Dr. Rita R. ColwellDistinguished Professor, University of Maryland College Park andJohns Hopkins University Bloomberg School of Public Health

Convergence Zone over the Convergence Zone over the Patagonian ShelfPatagonian ShelfDiscovery foments in the Discovery foments in the ““hyphenatedhyphenated””

zones of convergence.zones of convergence.

Source: Earth Observatory, NASA

The Space in Which We Work

Marine Technology

Marine Science•Living organism ID•Chemical separations•Physical parameters•Geology/hydrology•Boats, ships

Technology•Temperature control systems•Water supply•Cultivation technology•Chemical separation methods•Material processing equipment•Specialty materials supply

Marine BiotechnologyBiotechnology

Marine Biology

Biology•Genomics•Protein and enzyme purification•Metabolism studies•Foods and nutrients•Animal/human health

Courtesy of David G. Baden

Comparative GenomicsComparative Genomics

Sequence divergence

Differences between genera

Differences between species

Differences between isolates

Number of genomes sequenced in bacterial species*

Number of species % of the total Genomes/species297 86% 124 7% 213 4% 33 1% 42 1% 51 0.5% 71 0.5% 83 1% 9

______________________________________________*Data obtained from Genomes on Line database (www.genomesonline.org). •344 completed bacterial genomes listed as of September 1, 2006.

Welch, R.A. et al. (2002) Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 99: 17020-17024.

• Several comparative studies of multiple isolates of the same species have shown extensive genetic diversity in some species – in some cases ~ 25% of genes were unique to a single strain

• Escherichia coli (Fukiya et al., 2004)• Helicobacter pylori (Blaser et al., 2001)• Staphylococcus aureus (Fitzgerald et al., 2001)• Campylobacter jejuni (Dorrell et al., 2001)• Thermotoga maritima (Nesbo et al., 2002)

• Therefore, it is not possible to fully describe a particular species with a single genome sequence

The GBS pan-genome: defining the genetic diversity of a species

Each new genome identifies an average of 33 strain-specific genes

Genomic diversityGenomic diversity

genomic halogenomic halo(strain(strain--specific)specific)

non essentialnon essential

corecorecorecore

non essentialnon essential

CLOSED Pan-genome

Consistent with more isolatedlifestyle and limited access to

global microbial gene pool

OPEN Pan-genome

Consistent with colonization of multiple environments and opportunity for multiple

mechanisms of DNA exchange

G. Constantin de Magny

Vibrio cholerae

Small Chromosome Large Chromosome

Source: The Institute for Genomic Research

Strains used

• From diverse sources• Vibrio cholerae

– O1 El Tor: 13– O1 Classical: 1– O139: 1– Non O1/O139: 10

• Vibrio mimicus– 1 Environmental, 1 Clinical isolates

V. cholerae B33 O1 ElTor Ogawa Clin Mozambique 2004

V. cholerae MJ-1236 O1 hybrid biotype Clin Matlab 1994

V. cholerae MO10 O139 CT Madras 1992

Vibrio cholerae O1 biovar eltor str. N16961

V. cholerae RC9 O1 CT Kenya 1985

V. cholerae NCTC8457 O1 ElTor pre-Pan CT Clin Saudi Arabia 1910

V. cholerae MAK757 O1 ElTor Ogawa CT pre-7th Pan Celebes Is. 1937

V. cholerae 2740-80 O1 ElTor CT- Env Gulf 1980

V. cholerae O1 Classical 6th Pan CT Clin India 1965

V. cholerae V52 O37 CT Clin Sudan 1968

V. cholerae AM-19226 O39 CT- Clin Bangladesh 2001

V. cholerae MZO-2 O14 CT- Clin Bangladesh 2001

V. cholerae MZO-3 O37 CT- Clin Bangladesh 2001

V. cholerae 623-39

V. cholerae 1587 O12 Clin Lima 1994

V. cholerae TMA21 non-O1 CT Seawater Brazil 1982

V. cholerae V51 O141 Clin USA 1987

V. cholerae RC385 O135

V. cholerae VL426 V. albensis

0.005

Phylogeny of 19 V. cholerae based on 2.7 M base-long (2,620 ORFs) alignments

O1 El Tor/O139

O1 Classical/O37

Non-O1/O139

Mosaic genomic structure of V. choleraerevealed by comparative genomics

Chromosome I (2,961,149 bp, 2,742 ORFs)

RC9 O1 Ctx+ Kenya 2740-80 O1 ElTor Env USNCTC8457 O1 ElTor 1910B33 O1 ElTor Mzb MJ-1236 O1 hybrid biotype MO10 O139623-391587 O12VL426 albensis O395 O1 ClassicalAM-19226 O39 TMA21 non-O1 BrazilMAK757 O1 ElTor 1937 MZO-2 O14 MZO-3 O37 RC385 O135 Csp BayV51 O141 USV52 O37 Sudan

Chromosome II (1,072,315 bp, 1,093 ORFs)

RC9 O1 Ctx+ Kenya 2740-80 O1 ElTor Env USNCTC8457 O1 ElTor 1910B33 O1 ElTor Mzb MJ-1236 O1 hybrid biotype MO10 O139623-391587 O12VL426 albensis O395 O1 ClassicalAM-19226 O39 TMA21 non-O1 BrazilMAK757 O1 ElTor 1937 MZO-2 O14 MZO-3 O37 RC385 O135 Csp BayV51 O141 USV52 O37 Sudan

Missing ORFs in V. cholerae strains (Reference: N16961; cutoff = 70% DNA similarity)

Mosaic genomic structure revealed by comparative genomicsChromosome 1 (2742 genes)

Chromosome 2 (1093 genes)

Missing ORFs in V. cholerae strains (Reference: N16961; cutoff = 70% DNA similarity)

East Pacific Rise

Source: NSF/KDW, data from D. Chayes, LDEO, Columbia Univ.

Comparison of the Vibrio cholerae genome with the Vent Vibrio.

Blue are Amino Acid searches at >65% Similarity, Green are NA at >85% ID

0 500000 1000000 1500000 2000000 2500000 3000000

0 200000 400000 600000 800000 10000001200000

Chromosome II

Chromosome I

Ven

t Vib

rioV

ent V

ibrio

Ongoing Research• Definition of core-genome and pan-genome of

– The genus Vibrio– The species Vibrio cholerae– Vibrio cholerae O1

• Evolution of Vibrio cholerae (O1)• Discovery of SNP for large scale typing study• Use of genome data for targeted metagenomic survey

– Detection of variable regions to monitor the clonaldiversity of V. cholerae O1 in environments/hosts

• New highly sensitive molecular typing/monitoring methods can be used for studying impact of vaccines.

Sargasso Sea Sargasso Sea PicPic

Credit: Courtesy Mark L. Wells, School of Marine Sciences, University of Maine

CoccolithophoresCoccolithophores

Credit: Scanning electron micrographs by Vita Pariente at the Texas A&M University Electron Microscopy Center

Fosmid

LIbraries

Community Genomics Among Stratified Microbial Assemblages In the Ocean’s Interior

27 JANUARY 2006 VOL. 311 SCIENCE www.sciencemag.orgEdward F. DeLong, Christina M. Preston, Tracy Mincer, Virginia Rich, Steven J. Hallam Niels-Ulrik Frigaard, Asuncion Martinez, Matthew B. Sullivan, Robert Edwards, Beltran Rodriguez Brito, Sallie W. Chisholm, David M. Karl

10 m

70 m

200 m

130 m

500 m

770 m

4000 m

PHOTIC ZONE UNIQUE SEQUENCES

DEEP WATER UNIQUE SEQUENCES

PZ

DW

SYSTEMS BIOLOGY -> SYSTEMS ECOLOGY

SYSTEMSBIOLOGY

CELLS

POPULATIONS

COMMUNITIES

ECOSYSTEMS

From Eisenberg et al. , Nature 405:823-826

BiocomplexityBiocomplexity SpiralSpiral

Source: NSF/S. Raimo

Nacre Nacre

Sources: Nautilus: K.S. MatzInset (nacre): Zhiyong Tang, Oklahoma State University; NSF

Oyster ReefOyster ReefOyster Reef

Source: Shellfish Research Section, Marine Resources Research Institute, SCDNR

Sponge With DyeSponge With Dye

Source: © Sea Studios Foundation

Sea SquirtSea Squirt--AdultAdult

Source: DOE Joint Genome Institute © 2003

Sea SquirtSea Squirt--CompositeComposite

Source: DOE Joint Genome Institute © 2003

Source: John Delaney, University of Washington

Source: John Delaney, University of Washington School of Oceanography

Pompeii worm and DNA Pompeii worm and DNA SequenceSequence

DNA credit: University of Delaware College of Marine StudiesPompeii Worm: Craig Cary, University of Delaware College of Marine Studies

NanoshotNanoshot/Mark Wells/Mark Wells

Liposome Nanodevice

Credit: Courtesy Mark L. Wells, School of Marine Sciences, University of Maine

Source: Bonnie Bassler, Princeton University Note: bbassler@molbiol.princeton.eduNed Ruby and Margaret McFall Ngi, University of Hawaii

Quorum sensing diagramQuorum sensing diagram

Klebsiella pneumoniaeLactococcus lactisLeuconostoc oenosListeria monocytogenesNeisseria gonorrhoeaeNeisseria meningitidisPasteurella multocidaPorphyromonas gingivalisProteus mirabilisSalmonella paratyphiSalmonella typhiSalmonella typhimurium

Bacillus anthracisBacillus haloduransBacillus subtilisBorrelia burgdorferiCampylobacter jejuniClostridium acetobolyticumClostridium difficileClostridium perfringensDeinococcus radioduransEscherichia coliEnterococcus faecalisHaemophilus influenzaeHelicobacter pylori

Shewanella putrefaciensStaphylococcus aureusStaphylococcus epidermidisStreptococcus gordoniiStreptococcus mutansStreptococcus pneumoniaeStreptococcus pyogenesVibrio anguillarumVibrio choleraeVibrio harveyiVibrio vulnificusYersinia pestis

Source: Bonnie Bassler, Princeton University

Bacteria with the Signaling Molecule

BiofilmBiofilm

Credit: P. Dirckx 2003, courtesy Center for Biofilm Engineering, MSU-Bozeman

Credit: Centre for Marine Biofouling and Bioinnovation, University of New South Wales

Field trial, Furanones in Polymers (5 months)

Credit: Centre for Marine Biofouling and Bioinnovation, University of New South Wales

Furanone effect on bacterial clearance

Fur 30 treatmentSaline treatment

101066

101055

101044

101033

101022

101011

CFU

/lung

Mouse 1, 2, 3 Mouse 1, 2, 3 ……. etc. etc

Individual MiceSource: Mike Givskov

ConeshellsConeshells

Source: K.S. Matz; Collage: NSF/S.Raimo

The Tides of Red, orThe History of Toxin Discovery

<1600-- Spanish explorers report irritating essence from Florida red tide (ship’s log)

1948 --- Mistaken reports red tide gas may be world war I nerve gas (Press)1965 --- Reports of “fast” and “slow” acting toxins (McFarren, Sasner)1969 --- Phosphorylated toxin chemistry reported (Martin, Shimizu)1973 --- Isolation of hemolytic component from red tide (Martin and Padilla)1975 --- Neurotoxic component from red tide isolated (Baden, Risk, Shimizu)1980 --- Chemical structure of the first brevetoxin (Lin)1985 --- Multiple structures of brevetoxins, each with a demonstrated

specific toxicity (Shimizu, Baden)1990 --- Multiple brevetoxins, based on two structural backbones

(Baden, Shimizu)1995 --- Multiple brevetoxins & active metabolites/breakdown products

(Plakas, Ramsdell)2000 --- Multiple brevetoxins and metabolites, modulated by the polyether

antagonist brevenal (Bourdelais)2005 --- Multiple brevetoxins and metabolites, modulated by multiple

natural brevenal structures (Bourdelais) Courtesy of Daniel G. Baden

"Polyether Brevetoxins: What are they? What do they do? How do they do it?"

Diarrheic Shellfish Poisons(tethered polyethers)

Red Tide Brevetoxins(trans-syn polyethers)

Marine toxins disturb:•Nerve conduction•Immune function•DNA integrity•Memory and learning•Pulmonary function•Metabolism

Cone Shell Toxins(peptides)

Saxitoxin and Tetrodotoxin(low MW guanidiniums)

Courtesy of Daniel G. Baden

All Toxin Effects are as a ResultOf Specific Receptor-Toxin Interactions

Courtesy of Daniel G. Baden Poli et al; Risk et al; Baden et al; Trainer et al

Why does Karenia Make Brevetoxin?

1978 1985

•Lends competitive advantage (Have Gun, Will Travel)•Release nutrients (Feed Me, Feed Me)•Produced by a contaminant/symbiont (Detoxify)•Regulatory function (Produced for a Purpose)•Serendipity (Just Happens to Fit)

Therapeutic ToxicInconsequential

Courtesy of Daniel G. Baden

Synthetic PbTx-3-β-Naphthoate

Natural Brevetoxin

Baseline Brevenaltreated

Baseline Brevenaltreated

Time (min)

0 5 10 15 20 25 30 35 40 45

% R

eten

tion

of N

ebul

ized

Tc

99m

-Sul

fur C

ollo

id

40

50

60

70

80

90

100

110

Beta-Naphthoyl-PbTx-3 (n=4)Sterile Water (n=5)Albuterol (n=2)Brevenal (n=2)

Site-Directed Chemical Modification and

Activity Measurement:(Turn Harm Into Charm)

•Brevetoxins cause severe respiratory distress•Effects caused at picogram concentrations•Airway restriction increases over 250%

Natural Brevenal(AJB 6.0P)

•Both chemicals •counteract all toxin actions•treat symptoms of Cystic Fibrosis•treat mucociliary disorders•administered by nebulizer

•Three patents are pending•for seafood poisoning therapies•for treatment of mucociliary disease•general chemical structures of drugs

Bourdelais et al:

pg/ml

0 1 2

RL

(% in

crea

se)

0

50

100

150

200

250

G. breveG. breve + B-Naphthoyl-PbTx-3

pg/ml

0.01 0.1 1 10 100

RL

(% in

crea

se)

0

50

100

150

200

250

300

PbTx-2PbTx-2 + AJB 6.0P (100 pg/ml)PbTx-2 + AJB 6.0P (10 pg/ml)

Courtesy of Daniel G. Baden

Some Examples of Commercially Available Marine BioproductsPharmaceuticalsAra-A (acyclovir) Antiviral drug (infections) Marine sponge, Cryptotethya cryta

Ara-C (cytosar-U, cytarabine)

Anticancer drug (leukemia and non-Hodgkin’s lymphoma)

Marine sponge, Cryptotethya cryta

Molecular ProbesManoalide Phospholipase A2 inhibitor Marine sponge, Luffariella variabilis

Aequorin Bioluminescent Ca indicator Bioluminescent jellyfish, Aequora victoria

Green fluorescent protein Reporter gene Bioluminescent jellyfish, Aequora victoria

EnzymesVent and Deep Vent DNA poymerase (New England BioLabs)

Polymerase chain reaction enzyme

Deep-sea hydrothermal vent bacterium

Nutritional SupplementsFormulaid (MartekBiosciences)

Fatty acids used as additive in infant formula nutritional supplement

Marine microalga

PigmentPhycocrythrin Conjugated antibodies used in

ELISAs and flow cytometryRed algae

Cosmetic additivesResilience (Esteé Lauder) “Marine extract” additive Caribbean gorgonian, Pseudopterogorgia

elisabethaeSource: Adapted from Pomponi (1999)

Created by NSF/S. RaimoCredit: Adapted from a chart by Shirley A. Pomponi, Harbor Branch Oceanographic Institution

“Fundamental knowledge is still lacking in…commercialization of biomedical products and commercial application…to marine environmental problems…”

Source: NOAA

Prospecting partnershipsProspecting partnershipsIt would be a tragedy todestroy that which we have not yet even named, let alone understood.

Source: NOAA

Nature 2003Nature 2003

Left: Credit: Mike White/ Florida Keys National Marine SanctuaryRight: Credit: Thomas K. Gibson/ Florida Keys National Marine Sanctuary

QuoteQuoteWe have really just begun to explore the "full fathom five"

and beyond...

Source: NOAA