Transfer of Potential pseudomonas stutzeri genes
-
Upload
rob1323 -
Category
Health & Medicine
-
view
987 -
download
2
description
Transcript of Transfer of Potential pseudomonas stutzeri genes
![Page 1: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/1.jpg)
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
![Page 2: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/2.jpg)
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
![Page 3: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/3.jpg)
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.
![Page 4: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/4.jpg)
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
![Page 5: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/5.jpg)
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
![Page 6: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/6.jpg)
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
![Page 7: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/7.jpg)
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
![Page 8: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/8.jpg)
PSP (PARALYTIC SHELLFISH POISIONING)
Saxitoxin effects respiratory muscles
Symptoms: shortness of breath, dizziness, numbness of extremities. If not treated it can cause respiratory failure
![Page 9: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/9.jpg)
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
![Page 10: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/10.jpg)
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.
![Page 11: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/11.jpg)
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
![Page 12: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/12.jpg)
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
![Page 13: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/13.jpg)
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
![Page 14: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/14.jpg)
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. .
![Page 15: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/15.jpg)
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
![Page 16: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/16.jpg)
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.
![Page 17: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/17.jpg)
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”
![Page 18: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/18.jpg)
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
![Page 19: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/19.jpg)
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
![Page 20: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/20.jpg)
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
![Page 21: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/21.jpg)
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
![Page 22: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/22.jpg)
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
![Page 23: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/23.jpg)
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. .
![Page 24: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/24.jpg)
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
![Page 25: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/25.jpg)
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
![Page 26: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/26.jpg)
METHOD ONEMETHOD ONE
Molecular approach
Design PCR primers to amplify the sxt genes in Pseudomonas stutzeri marine isolate.
![Page 27: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/27.jpg)
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
![Page 28: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/28.jpg)
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
![Page 29: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/29.jpg)
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
![Page 30: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/30.jpg)
Preliminary Results Preliminary Results
![Page 31: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/31.jpg)
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?
![Page 32: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/32.jpg)
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
![Page 33: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/33.jpg)
Preliminary Results
E-value
Similarity between the genes
Lower E-value
Good match
![Page 34: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/34.jpg)
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
![Page 35: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/35.jpg)
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
![Page 36: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/36.jpg)
5 out of 12 P. stutzeri genes did not have a close BLAST match with cyanobacterial STX enzymes
Preliminary Results
![Page 37: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/37.jpg)
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
![Page 38: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/38.jpg)
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
![Page 39: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/39.jpg)
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
![Page 40: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/40.jpg)
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
![Page 41: Transfer of Potential pseudomonas stutzeri genes](https://reader033.fdocuments.in/reader033/viewer/2022060117/55855a96d8b42a970b8b4578/html5/thumbnails/41.jpg)
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