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 … · – the...
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・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.
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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
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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
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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
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Comparative GenomicsComparative Genomics
Sequence divergence
Differences between genera
Differences between species
Differences between isolates
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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.
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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.
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• 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
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The GBS pan-genome: defining the genetic diversity of a species
Each new genome identifies an average of 33 strain-specific genes
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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
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G. Constantin de Magny
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Vibrio cholerae
Small Chromosome Large Chromosome
Source: The Institute for Genomic Research
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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
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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
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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)
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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)
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East Pacific Rise
Source: NSF/KDW, data from D. Chayes, LDEO, Columbia Univ.
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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
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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.
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Sargasso Sea Sargasso Sea PicPic
Credit: Courtesy Mark L. Wells, School of Marine Sciences, University of Maine
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CoccolithophoresCoccolithophores
Credit: Scanning electron micrographs by Vita Pariente at the Texas A&M University Electron Microscopy Center
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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
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10 m
70 m
200 m
130 m
500 m
770 m
4000 m
PHOTIC ZONE UNIQUE SEQUENCES
DEEP WATER UNIQUE SEQUENCES
PZ
DW
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SYSTEMS BIOLOGY -> SYSTEMS ECOLOGY
SYSTEMSBIOLOGY
CELLS
POPULATIONS
COMMUNITIES
ECOSYSTEMS
From Eisenberg et al. , Nature 405:823-826
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BiocomplexityBiocomplexity SpiralSpiral
Source: NSF/S. Raimo
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Nacre Nacre
Sources: Nautilus: K.S. MatzInset (nacre): Zhiyong Tang, Oklahoma State University; NSF
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Oyster ReefOyster ReefOyster Reef
Source: Shellfish Research Section, Marine Resources Research Institute, SCDNR
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Sponge With DyeSponge With Dye
Source: © Sea Studios Foundation
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Sea SquirtSea Squirt--AdultAdult
Source: DOE Joint Genome Institute © 2003
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Sea SquirtSea Squirt--CompositeComposite
Source: DOE Joint Genome Institute © 2003
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Source: John Delaney, University of Washington
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Source: John Delaney, University of Washington School of Oceanography
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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
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NanoshotNanoshot/Mark Wells/Mark Wells
Liposome Nanodevice
Credit: Courtesy Mark L. Wells, School of Marine Sciences, University of Maine
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Source: Bonnie Bassler, Princeton University Note: [email protected] Ruby and Margaret McFall Ngi, University of Hawaii
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Quorum sensing diagramQuorum sensing diagram
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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
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BiofilmBiofilm
Credit: P. Dirckx 2003, courtesy Center for Biofilm Engineering, MSU-Bozeman
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Credit: Centre for Marine Biofouling and Bioinnovation, University of New South Wales
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Field trial, Furanones in Polymers (5 months)
Credit: Centre for Marine Biofouling and Bioinnovation, University of New South Wales
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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
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ConeshellsConeshells
Source: K.S. Matz; Collage: NSF/S.Raimo
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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
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"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
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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
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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
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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
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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
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“Fundamental knowledge is still lacking in…commercialization of biomedical products and commercial application…to marine environmental problems…”
Source: NOAA
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Prospecting partnershipsProspecting partnershipsIt would be a tragedy todestroy that which we have not yet even named, let alone understood.
Source: NOAA
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Nature 2003Nature 2003
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Left: Credit: Mike White/ Florida Keys National Marine SanctuaryRight: Credit: Thomas K. Gibson/ Florida Keys National Marine Sanctuary
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QuoteQuoteWe have really just begun to explore the "full fathom five"
and beyond...
Source: NOAA
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