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