1

The use of DGGE analyses to exploreThe use of DGGE analyses to exploreeasterneastern

Mediterranean and Red Sea marineMediterranean and Red Sea marinepicophytoplanktonpicophytoplankton

assemblagesassemblages

Gil Gil ZeidnerZeidner and and OdedOded BéjàBéjàDepartment of Biology, Department of Biology, TechnionTechnion-Israel Institute of-Israel Institute of

Technology, Haifa 32000, Israel.Technology, Haifa 32000, Israel.

Environmental Microbiology, 2004, Vol. 6:528-534Environmental Microbiology, 2004, Vol. 6:528-534

Presented by Christine Bevacqua

Background: Marine Background: Marine photosythesisphotosythesis

Most photosynthetic production in openMost photosynthetic production in openocean performed by cells smaller than 2-3ocean performed by cells smaller than 2-3µµm (m (picoplanktonpicoplankton))

CynobacteriaCynobacteria, eukaryotes containing, eukaryotes containingchlorophyll pigmentschlorophyll pigments

PhotosynthicPhotosynthic apparatus: apparatus: photosystemphotosystem 1 1and 2and 2

PhotosystemPhotosystem II II Membrane embeddedMembrane embedded

protein complexprotein complex D1 and D2 proteinsD1 and D2 proteins

form reaction centerform reaction centerdimerdimer

D1 coded by D1 coded by psbpsbAAgenegene

psbpsbAA well conserved well conservedin in cyanobacteriacyanobacteria and andeukaryoteseukaryotes

www.life.uiuc.edu/govindjee/paper/gov.html

2

Culture-independent Molecular MethodsCulture-independent Molecular Methods

Most marine diversity found throughMost marine diversity found throughculture-independent surveysculture-independent surveys

Largely expanded knowledge of microbialLargely expanded knowledge of microbialdiversitydiversity

Denaturing Gradient Gel ElectrophoresisDenaturing Gradient Gel Electrophoresis(DGGE)(DGGE)

DGGEDGGE

Separate DNA based on Separate DNA based on ““melting pointmelting point”” in inpolyacrylamidepolyacrylamide gel. gel.

Uses PCR amplificationUses PCR amplification Good for high-throughput studiesGood for high-throughput studies Often based on 16s Often based on 16s rRNArRNA genes genes Recent studies focusing on functionalRecent studies focusing on functional

protein-coding genesprotein-coding genes

Use of Use of psbpsbAA gene gene

Photosynthetic protein coding gene (D1)Photosynthetic protein coding gene (D1)

Enables the study to focus onEnables the study to focus onphotosynthetic microorganismsphotosynthetic microorganisms

Recently used by authors to search forRecently used by authors to search formolecular diversity among molecular diversity among planktonicplanktonicmicroorganisms- microorganisms- phylogeneticphylogenetic marker marker

3

Primer Performance CheckPrimer Performance Check

DGGE method testedDGGE method testedwith previouslywith previouslyisolated isolated psbpsbAA clones clones

PCR known to havePCR known to havebiases- will it work?biases- will it work?

All species in a knownAll species in a knownmix detectedmix detected

Red and Mediterranean SeaRed and Mediterranean SeaSamplesSamples

♦

♦

♦

http://encarta.msn.com

Red Sea ResultsRed Sea Results

RED132: Outside gulfRED132: Outside gulf CyanobacteriaCyanobacteria No change betweenNo change between

depthsdepths RED122: Gulf of RED122: Gulf of AqabaAqaba

PicoeukaryoticPicoeukaryotic algae algae 8, 15, 13 only at 60 and8, 15, 13 only at 60 and

150 m150 m 10 dominates 60 m10 dominates 60 m

4

Red Sea ResultsRed Sea Results

Gulf of Gulf of AqabaAqaba composition agrees with composition agrees withprevious results (previous results (ZeidnerZeidner et. al 2003) et. al 2003)obtained with obtained with psbpsbAA libraries libraries

ChlorophytaChlorophyta sequences at 122, absent at sequences at 122, absent at132. Also in agreement with previous132. Also in agreement with previouswork (work (BiegalaBiegala et al. 2003) et al. 2003)

Mediterranean ResultsMediterranean Results

CyanobacteriaCyanobacteria dominate dominatephoticphotic zone zone

Mainly Mainly ProchlorococcusProchlorococcus 1 dominates1 dominates 8, 9 only at 150 m8, 9 only at 150 m

SynechococcusSynechococcus present at present atall depths, higher at 100 mall depths, higher at 100 m

SynechococcusSynechococcus SynechococcusSynechococcus found are related but different from found are related but different from

previously retrieved genotypes at same stationpreviously retrieved genotypes at same station

Sequences also related to new Sequences also related to new psbpsbAA sequence found on sequence found onvirus genomevirus genome

SynechococcusSynechococcus WH8102 genome contains 4 copies ofWH8102 genome contains 4 copies ofpsbpsbAA gene gene

Not possible to say if bands are coming from theseNot possible to say if bands are coming from thesesourcessources

Possible primer biases?Possible primer biases?

5

Cyanobacteria psbA sequences Eukaryotic psbA sequences

More agreementsMore agreements

Low light (LL) and High light (HL)Low light (LL) and High light (HL)ProchlorococcusProchlorococcus observed and separatedobserved and separatedon treeson trees

Red8, red13: fall with LL, found at 60-150 mRed8, red13: fall with LL, found at 60-150 m Same with med8, med9Same with med8, med9 Also HL-like Also HL-like ProchlorococcusProchlorococcus found at allfound at all

depths similar to previous observationsdepths similar to previous observations(West et al 2001).(West et al 2001).

Conclusion:Conclusion:DGGE is a good tool for quicklyDGGE is a good tool for quickly

accessing and assessing compositionaccessing and assessing compositionof different phytoplankton assemblagesof different phytoplankton assemblagesin marine ecosystems and should helpin marine ecosystems and should helpin revealing the composition of thesein revealing the composition of these

assemblages worldwideassemblages worldwide

6

Next StepsNext Steps

What are the functions of theseWhat are the functions of thesephytoplankton? How do they fit into thephytoplankton? How do they fit into therest of the ecosystem?rest of the ecosystem?

Further study the Further study the SynechococcusSynechococcus that thatwere found in this study and the previouswere found in this study and the previousone?one?

Survey more areas of the ocean in thisSurvey more areas of the ocean in thiswayway