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In this research of saxitoxin bacterial production bioinformatics will be utilized to identify sxt coding genes within pseudomonas stutzeri strain A1501. PCR will then be employed in the isolation of sxt genes from pseudomonas stutzeri. The isolated genes will be transformed into Ecoli cultures.
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Building Genetic Foundations Building Genetic Foundations in the saxitoxin Production in the saxitoxin Production
of of Pseudomonas stutzeriPseudomonas stutzeri
A Research ProposalA Research ProposalGabrielle RobertsGabrielle Roberts
TABLE OF CONTENTSTABLE OF CONTENTS
I.I. ALGAE BLOOMSALGAE BLOOMS
II.II. BACTERIAL PRODUCTION OF BACTERIAL PRODUCTION OF SAXITOXIN: A CONTROVERISAL SAXITOXIN: A CONTROVERISAL TOPIC TOPIC
III.III. SAXITOXIN A NEUROTOXINSAXITOXIN A NEUROTOXIN
IV.IV. CHEMICAL & MOLECULAR SIDES CHEMICAL & MOLECULAR SIDES OF SAXITOXIN SYNTHESIS OF SAXITOXIN SYNTHESIS
V.V. METHODSMETHODS
VI.VI. PRELIMINARY RESULTSPRELIMINARY RESULTS
OVERVIEW OF BLOOMSOVERVIEW OF BLOOMS Saxitoxin is a potent neurotoxin that can Saxitoxin is a potent neurotoxin that can
be found within Harmful Algae Blooms of be found within Harmful Algae Blooms of cyanobacteria and dinoflagellates.cyanobacteria and dinoflagellates.
Harmful Algae Blooms
Bloom: “population explosions” or large populations of algae within marine and fresh water environments.
HAB “red tide”
Blooms of toxic dinoflagellates in marine environment
Green bloom of blue-green algae cyanobacteria in fresh water
CauseCause: Eutrophication events: addition of nitrogen and phosphorus from agricultural run offs or sewage leakage can cause an increase in algae population number
Description of algae bloomsDescription of algae blooms: - Blooms can be toxic or nontoxic- Involve cyanobacteria, dinoflagellates - Have detrimental effects on
environments and human population
Impacts of non-toxic/toxic blooms
Large Populations of algae can prevent light from reaching lower depths
Submerged plants growing at the bottom don’t have access to light. Plants get rid of nitrogen/phosphorus pollutants.
Anoxia: blooms cause
oxygen depletion of marine and fresh water environments
Littoral zone: area where plants grow in pond or lakes
http://www.dnr.state.mn.us/shorelandmgmt/apg/wheregrow.html
Impacts of toxic blooms“the food pyramid”
Consumption of saxitoxin contaminated seafood can have devastating effects
Humans
SECONDARY CONSUMERS
Fish
PRIMARY CONSUMERSFilter feeders
Shrimp “have natural saxitoxin resistance”
PRIMARY PRODUCERSPhotoautotrophs
Dinoflagellates cyanobacteria
PSP (PARALYTIC SHELLFISH POISIONING)
Saxitoxin effects respiratory muscles
Symptoms: shortness of breath, dizziness, numbness of extremities. If not treated it can cause respiratory failure
QUESTION:
How do dinoflagellates produce saxitoxin?
Sxanning electron micrograph of dinoflagellate: Gymnodium
DAB dinoflagellate asscoiated bacteria
Phylum: Cytophage-Flavobacteroides and Proteobacteria
Live in Alexandrium and Gymnodinium dinoflagellates
Bacterial saxitoxin production a controversial subject…
Past 1995-1996 studies affermed bacterial paralytic saxitoxin production using bacteria isolated from Alexandrium lusitanicum and Gymnodium Catenatum
Current 2003 research findings have not found evidence linking bacteria to the production of paralytic saxitoxin
Incorrect identification of paralytic saxitoxin within dinoflagellate producing bacteria using methods such as HPLC.
Evidence of saxitoxin bacteria Evidence of saxitoxin bacteria productionproduction
Evidence: Horizontal gene transfersEvidence: Horizontal gene transfers
Bacteria transferred saxitoxin gene into Bacteria transferred saxitoxin gene into cyanobacteria. cyanobacteria.
Result: saxitoxin producing cyanobacteria Result: saxitoxin producing cyanobacteria
Bacteria
Bacteria
cyanobacteria
Evidence of saxitoxin bacteria Evidence of saxitoxin bacteria production…production…
Sxt genes have been characterized within Sxt genes have been characterized within saxitoxin producing cyanobacteria. saxitoxin producing cyanobacteria.
Phylogenetic analysis show that Phylogenetic analysis show that some cyanobacteria stx genes have bacteria origins.
Moustafa, A., Loram, J. E., Hackett, J. D., Anderson, D. M., Plumley, F. G., Bhattacharya, D. (2009). Origins of Saxitoxin Biosynthetic Genes in Cyanobacteria. Plos One, 4 (6). Retrieved from: www.plosone.org
SXTA: Polyketide synthaseSXTA: Polyketide synthaseFirst enzyme in biosynthesis. Has 4 domains.
1. Acyl-CoA/N-acyltransferase
2. Phosphopantetheine binding site
Aminotransferase classI and II
Proteobacterium Acintobacterium
STX+ Cyanobacteria
Moustafa, A., Loram, J. E., Hackett, J. D., Anderson, D. M., Plumley, F. G., Bhattacharya, D. (2009). Origins of Saxitoxin Biosynthetic Genes in Cyanobacteria. Plos One, 4 (6). Retrieved from: www.plosone.org
SxtA
SAXITOXIN MODE OF ACTIONSAXITOXIN MODE OF ACTION Affects the Affects the sodium gated channels of sodium gated channels of
nerve cells. nerve cells. Prevents generation of neural impulses by Prevents generation of neural impulses by
inhibiting depolarization eventsinhibiting depolarization events. .
Repolarization
Rest State
Passer, M.W. & Smith, R. E. (2004). Psychology the Science of Mind and Behavior: Second Edition. New York: McGraw Hill.
Depolarization
Repolarization
SAXITOXIN BINDING SAXITOXIN BINDING The sodium voltage gated channel The sodium voltage gated channel 260kDa a-subunit and 33-36kDa beta
subunit
The a-subunit is made up of 4 transmembrane domains.
Each domain has S1-S6 segments
a-subunit
Zimmer, R. K., & Ferrer, R. P. (2007). Neuroecology, Chemical Defense and the Keystone Species Concept. Biol. Bull., 213, 208-225.
a-subunit Ion conducting pore The pore loop between the S5 and S6 segments
1. Na+ enter
2. Is the site where saxitoxin binds
“Inner ring pore loop”
SAXTITOXIN CHEMICAL SAXTITOXIN CHEMICAL STRUCTURESTRUCTURE
The guanidine, hydroxyl and carbamoyl groups of saxitoxin bind to the voltage gated sodium channel’s inner ring pore loop
SAXTITOXIN CHEMICAL SAXTITOXIN CHEMICAL SYNTHESISSYNTHESIS
Chemical synthesis of saxitoxin Chemical synthesis of saxitoxin involves 9 steps involves 9 steps
use several enzymes and co-factorsuse several enzymes and co-factors
Transfer of guanidino group to intermediate
Claisen condensation of arginine and acetate
Kellman, R., Mihali, T. K., Jeon, Y. J., Pickford, R., Pomati, F., & Neilan, B. A. (2008). Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria. Applied and Environmental Microbiology, 74, No.13, 4044-4053.
carbamoylation
hydroxylation
SAXTITOXIN MOLECULAR SAXTITOXIN MOLECULAR SYNTHESISSYNTHESIS
SxtG
Guanidino transfer
SxtB
SxtD
Cyclization
Methylation
Epoxidation
SxtSSxtSopening
SxtU Reduction
Carbamoylation
SxtH/TSxtWSxtV
Hydroxylation
SxtI
Claisen condensation: acetate to arginine
GOAL: TO PROVE THAT
PSEUDOMONAS STUTZERI CAN SYNTHESIZE SAXITOXIN
- Increase our understanding of dinoflagellate saxitoxin synthesis
- Develop methods to reduce the occurrence of Red Tide
- Help fishing industries, reduce PSP fatalities
OBJECTIVE: OBJECTIVE:
. . To uncover evidence of the bacterial saxitoxin To uncover evidence of the bacterial saxitoxin production within production within Pseudomonas stutzeriPseudomonas stutzeri marine strain through the use of marine strain through the use of bioinformatics and and molecular biologymolecular biology. .
THE MODEL ORGANISMSTHE MODEL ORGANISMS Pseudomonas stutzeriPseudomonas stutzeri strains strains
Pseudomonas stutzeriPseudomonas stutzeri A1501 (sxt-) A1501 (sxt-)
Pseudomonas stutzeriPseudomonas stutzeri marine strain (sxt+) marine strain (sxt+)
CyanobacteriaCyanobacteria
Cylindrospermopsis raciborskiiCylindrospermopsis raciborskii T3 T3 (sxt+)(sxt+)
Saxitoxin genes have been sequencedSaxitoxin genes have been sequenced
Question: Why use bioinformatics? Question: Why use bioinformatics?
•Search for cyanobacteria saxitoxin genes Search for cyanobacteria saxitoxin genes within within Pseudomonas stutzeriPseudomonas stutzeri (sxt-)(sxt-) Conduct Conduct BLAST BLAST search for cyanobacteria saxitoxin genes in search for cyanobacteria saxitoxin genes in Pseudomonas stutzeriPseudomonas stutzeri (sxt-) (sxt-)
•Phylogenetic analysis of saxitoxin producing Phylogenetic analysis of saxitoxin producing enzymes in cyanobacteria and enzymes in cyanobacteria and Pseudomonas Pseudomonas stutzeristutzeri (sxt-) (sxt-)
Use Use CLUSTALW programCLUSTALW program to conduct a phylogenetic analysis of saxitoxin to conduct a phylogenetic analysis of saxitoxin enzymes in enzymes in Pseudomonas stutzeriPseudomonas stutzeri A1501 and cyanobacteria A1501 and cyanobacteria Cylindrospermopsis raciborskiiCylindrospermopsis raciborskii T3 T3
METHOD ONEMETHOD ONE
Molecular approach
Design PCR primers to amplify the sxt genes in Pseudomonas stutzeri marine isolate.
METHOD ONEMETHOD ONE2. Insert the saxitoxin DNA within competent E. coli cells
via bacterial transformation. - finding suitable vector that is able to express all
amplified gene sequences * BAC (bacterial artificial chromosome)
3. Confirm bacterial saxitoxin production using HPLC (high preformance liquid chromatography)
Identify saxitoxin in E-coli cultures
Question: Question:
Why Why Pseudomonas stutzeriPseudomonas stutzeri? ?
Pseudomonas stutzeriPseudomonas stutzeri is a dinoflagellate is a dinoflagellate associated bacteria that is commonly isolated from associated bacteria that is commonly isolated from dinoflagellatesdinoflagellates
Question:Question:
Why two strains of Why two strains of Pseudomonas stutzeriPseudomonas stutzeri 1. Pseudomonas stutzeri A1501 is a rice strain that does not make
saxitoxin. But it is closely related to the Pseudomonas strutzeri marine strain
2. The genome of saxitoxin producing Pseudomonas stutzeri has not been sequenced
METHOD TWO METHOD TWO 1. Commercially sequence DNA isolated from
Pseudomonas stutzeri marine strain
2. Conduct search for genes coding for saxitoxin enzymes
Ambry Genetics
Preliminary Results Preliminary Results
Preliminary Results
Analysis of the P.sutzeri A1501 hypothetical
enzymes show that the Pseudomonas stutzeri A1501 genes:
1. Do not form an open reading frame gene cluster2. Are instead separated by great distances and some are on
different strands.
Positive strand
Negative strand
13 million base pairs apart
3’ 5’
5’ 3’
5’ 3’
sxtH sxtU
sxtI
Where are the Pseudomonas stutzeri A1501 genes located?
Preliminary Results CLUSTAL W Phylogenetic Analysis
Cylindrospermopsis raciborskii T3 saxitoxin enzymes
Pseudomonas stutzeri A1501 BLAST results
SxtU
Short-chain dehydrogenase
Cylindrospermopsis raciborskii T3
Pseudomonas stutzeri A1501Common ancestor
All C. Raciborskii T3 and Pseudomonas stutzeri A1501 enzymes are evolutionary related
HYPOTHETICAL PHYLOGENETIC TREE
Preliminary Results
E-value
Similarity between the genes
Lower E-value
Good match
Preliminary Results
Good
E-values
Carbamoyl Transferase
7e-88
Short-chain dehydrogenase
3e-40
8-amino-7-oxononanoate
9e-44
Closest Blast Match in P. stutzeri A1501
STX Enzyme in C.raciborskii T3
sxtI
sxtU
sxtA
Carbamoyl transferase E-value: 7e-88
Exceptional E-value is evidence of horizontal gene transfer between Cylindrospermopsis raciborskii T3 and Pseudomonas stutzeri A1501
Pseudomonas stutzeri A1501
5 out of 12 P. stutzeri genes did not have a close BLAST match with cyanobacterial STX enzymes
Preliminary Results
Preliminary Results
Iron sulfur Protein 0.001
Sterol desaturase 0.037
Tyosine decarboxylaseputative
0.063
Closest Blast Match in P. stutzeri A1501
STX Enzyme in C.raciborskii T3
sxtW
sxtD
sxtJ
sxtB Hypothetical proteinPST_2386
0.14
Chemotaxis ProteinCheV
sxtG 1.9
E-value
Preliminary Results
Moderate
E-values
Proteins are found in all bacteria
Some have the same function as the cyanobacteria saxitoxin enzyme
Short-chain dehydrogenase
3e-40
Protein involved in the biosynthesis of mitomycin antibiotics/polyketide fumonisin
2e-04
Closest Blast Match in P. stutzeri A1501
STX Enzyme in C.raciborskii T3
sxtU
sxtS
sxtH Benzoate dioxygenase, alpha subunit
2e-o4
8-amino-7-oxononanoate
sxtA 9e-44
E-value
1e-o4sxtI
we designed primers to match the Pseudomonas stutzeri A1501 saxitoxin enzymes that had a good-moderate
E-values
we designed primers for the cyanobacterial saxitoxin enzymes that had a bad
high-value
Currently… Use PCR to amplify P.stutzeri rice strain
STX genes/ cyanobacterial STX genes from marine P.stutzeri strain.
Commercially sequence genes from the Pseudomonas stutzeri marine strain
using Ambry Genetics
ACKNOWLEDGEMENTSACKNOWLEDGEMENTSSpecial Thanks to: Special Thanks to: Committee membersCommittee members Dr. MarshDr. Marsh Dr. MolinaDr. Molina Dr. Morin Dr. Morin
Research funding: Research funding: ARCC program ARCC program