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By Robert Eagen &By Robert Eagen &HenokHenok WoldeabWoldeab

New Insights into the Lifestyle of theNew Insights into the Lifestyle of theCold-Loving SM1 Euryarchaeon: NaturalCold-Loving SM1 Euryarchaeon: NaturalGrowth as a Growth as a MonospeciesMonospecies BiofilmBiofilm in the in the

SubsurfaceSubsurface

HennebergerHenneberger et al. Applied & Environmental Microbiology et al. Applied & Environmental Microbiology Jan2006, Vol.Jan2006, Vol.72 Issue 1, p192-19972 Issue 1, p192-199

Background: SM1 EuryarchaeonBackground: SM1 Euryarchaeon

Cold Cold SulfidicSulfidic Springs are known sites of high bioactivity Springs are known sites of high bioactivity Sulfur streamlets are home to a unique microbial communitySulfur streamlets are home to a unique microbial community

called SM1 Euryarchaeon, which forms string-of-pearlscalled SM1 Euryarchaeon, which forms string-of-pearlscommunities connected by filamentous bacteria.communities connected by filamentous bacteria.

The SM1 Euryarchaeon always made up the interior of theThe SM1 Euryarchaeon always made up the interior of thepearl while the type of filamentous bacteria composing thepearl while the type of filamentous bacteria composing theexterior changed based on location and conditions.exterior changed based on location and conditions.

Relevant Studies: Streamlets ofRelevant Studies: Streamlets ofSippenauerSippenauer Moor Moor

A string of whitish pearl connected by thin white threads wereA string of whitish pearl connected by thin white threads werefound in the streamletsfound in the streamlets

SM1 SM1 euryarchaeoneuryarchaeon was shown to compose most of the pearl was shown to compose most of the pearl’’ssinterior while the filamentous sulfur oxidizing interior while the filamentous sulfur oxidizing ThiothrixThiothrix sp. sp.composed the exterior.composed the exterior.

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Relevant Studies: Streamlets ofRelevant Studies: Streamlets ofIslingerIslinger MühlbachMühlbach

A second type of string-of-pearls community wasA second type of string-of-pearls community wasdiscovered under low in situ oxygen concentrations.discovered under low in situ oxygen concentrations.

SM1 SM1 euryarchaeoneuryarchaeon was shown to compose most of the was shown to compose most of thepearlpearl’’s interior while the filamentous IMB1s interior while the filamentous IMB1εε––proteobacteriumproteobacterium composed the exterior. composed the exterior.

Coexistence of SM1 Coexistence of SM1 euryarchaeoneuryarchaeon with specific bacterial with specific bacterialpartners suggested a symbiotic relationship with somepartners suggested a symbiotic relationship with sometype of sulfur cycle or nutrient exchange within eachtype of sulfur cycle or nutrient exchange within eachpearl.pearl.

Is this always the case?Is this always the case?

Cultivation TechniqueCultivation Technique

A new cultivation technique was used in aA new cultivation technique was used in astudy at study at IslingerIslinger MühlbachMühlbach where wherepolyethylene nets were placed in coldpolyethylene nets were placed in coldsulfidicsulfidic streamlets. streamlets.

Found whitish pearls on the nets growingFound whitish pearls on the nets growingwithin 2 to 3 days with SM1 Euryarchaeonwithin 2 to 3 days with SM1 Euryarchaeondominating the interior of the pearlsdominating the interior of the pearls

Unique AppendageUnique Appendage

Analysis of the pearls foundAnalysis of the pearls foundthat the SM1 Euryarchaeonthat the SM1 Euryarchaeonhad a unique cell surfacehad a unique cell surfaceappendage described asappendage described asnanonano-grappling hooks and-grappling hooks andnanonano-barbed-wire. The-barbed-wire. Theappendages were namedappendages were namedhamihami..

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Current ExperimentCurrent Experiment

This is a follow upThis is a follow upexperiment to recentexperiment to recentmicrobial surveys of coldmicrobial surveys of coldsulfidicsulfidic springs in Bavaria, springs in Bavaria,Germany.Germany.

All previously conductedAll previously conductedexperiments have dealt withexperiments have dealt withthe study of SM1the study of SM1Euryarchaeon found on theEuryarchaeon found on thesurface of these sulfursurface of these sulfursprings.springs.

ObjectivesObjectives

To investigate the possible occurrence ofTo investigate the possible occurrence ofthe SM1 the SM1 euryarchaeoneuryarchaeon in subsurface water in subsurface water

To test the ability of a newly designed inTo test the ability of a newly designed insitu situ biofilmbiofilm trapping system in the trapping system in thecollection of large quantities of microbialcollection of large quantities of microbialcommunities.communities.

Why Is This Important?Why Is This Important?

Increases our knowledge of the biologyIncreases our knowledge of the biologyand ecological significance of and ecological significance of archaeaarchaea in intheir environments.their environments.

EuryarchaeonEuryarchaeon is suspected of having a is suspected of having asulfate reducing metabolism.sulfate reducing metabolism.

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In situ In situ biofilmbiofilm trapping system trapping system

Nets of polyethylene treated with 70%Nets of polyethylene treated with 70%ethanol (ethanol (vol/volvol/vol))

Twelve circular frames were arranged in aTwelve circular frames were arranged in aplastic tube with each frame containingplastic tube with each frame containingfive polyethylene nets and then placedfive polyethylene nets and then placedinto a drill hole to allow spring water tointo a drill hole to allow spring water topass through the netspass through the nets

In-situ In-situ biofilmbiofilm trapping system trapping system

Other Techniques UsedOther Techniques Used

Phase-contrast, Phase-contrast, epifluorescenceepifluorescence, electron, electronmicroscopy, FISH, microscopy, FISH, oligonucleotideoligonucleotide probes, probes,and and phylogeneticphylogenetic analyses using PCR analyses using PCRamplification, RFLP, and sequencing.amplification, RFLP, and sequencing.

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Results:Results:In situ attachment and growth experiments with microbialIn situ attachment and growth experiments with microbial

communities on polyethylene netscommunities on polyethylene nets

The polyethylene nets were exposed to bothThe polyethylene nets were exposed to bothspring and streamletspring and streamlet

Rapid growth (in 3 to 4 days) of string of pearlsRapid growth (in 3 to 4 days) of string of pearlscommunities in the oxygen containing zones atcommunities in the oxygen containing zones atdifferent sites along the streamlets.different sites along the streamlets.

No growth of string of pearls in the drill hole andNo growth of string of pearls in the drill hole andthe area 10 cm surrounding it (oxygen poor)the area 10 cm surrounding it (oxygen poor)

In these areas, they found milky slime likeIn these areas, they found milky slime likeclumps attaching to the polyethylene netsclumps attaching to the polyethylene nets

Identification of biomaterialIdentification of biomaterial

Phase contrast microscopy showed small Phase contrast microscopy showed small coccoidcoccoid shaped microbes shaped microbesthat were arranged three dimensionally in a similar pattern to SM1that were arranged three dimensionally in a similar pattern to SM1euryarchaeoneuryarchaeon in the string of pearl communities. in the string of pearl communities.

FISH indicated that over 95% of microbial population were FISH indicated that over 95% of microbial population were coccoidcoccoidarchaealarchaeal cells and all the cells and all the archaealarchaeal tested positive for SM1-specific tested positive for SM1-specificeuryarchaealeuryarchaeal probes probes

These cell were confirmed as predominately SM1 These cell were confirmed as predominately SM1 euryarchaealeuryarchaeal cells cellsby RFLP analysis, 16S by RFLP analysis, 16S rRNArRNA gene sequence analysis, and gene sequence analysis, andsequencing of the 16S-23S sequencing of the 16S-23S rRNArRNA intergenicintergenic spacer region. spacer region.

Sequences were shown to be identical to the SM1 Sequences were shown to be identical to the SM1 euryarchaealeuryarchaealsequences of the strings of pearls communitiessequences of the strings of pearls communities

These These archaealarchaeal phylogeneticphylogenetic studies discovered the first studies discovered the first archaealarchaealmonospeciesmonospecies composed of SM1 composed of SM1 euryarchaoneuryarchaon in nature in nature

Quantitative collection ofQuantitative collection ofeuryarchaealeuryarchaeal SM1 SM1 biofilmsbiofilms

Using the Using the biofilmbiofilm trapping system, they were able to harvest trapping system, they were able to harvestin a quick and reproducible manner about 1.5 to 2.0 g (wetin a quick and reproducible manner about 1.5 to 2.0 g (wetweight) of weight) of euryarchaealeuryarchaeal SM1 SM1 biofilmbiofilm within 4 days, which is within 4 days, which isequivalent to 7 x 10equivalent to 7 x 1099 to 9 x 10 to 9 x 1099 cells. cells.

After just 10 min of exposure to polyethylene net, visibleAfter just 10 min of exposure to polyethylene net, visibleamounts of amounts of biofilmbiofilm were harvested indicating were harvested indicating biofilmsbiofilms were werewashed from deeper underground layers.washed from deeper underground layers.

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Physiological and biochemicalPhysiological and biochemicalinvestigation of subsurface-derivedinvestigation of subsurface-derived

SM1 SM1 biofilmsbiofilms

95% of SM1 95% of SM1 euryarchaealeuryarchaeal cells in cells in biofilmbiofilm from subsurface were from subsurface wereviableviable

pH of interior of pH of interior of biofilmbiofilm was between 7 and 7.5 was between 7 and 7.5 Indicated presence of carbohydrates and proteins in a total ratio ofIndicated presence of carbohydrates and proteins in a total ratio of

1:1.51:1.5

Genetic Studies/ Genetic Studies/ UltrastructuralUltrastructuralstudiesstudies

Detection of a single band following PFGE indicatedDetection of a single band following PFGE indicateda total genome size of 3 a total genome size of 3 MbpMbp for SM1 for SM1 euryarchaeoneuryarchaeon..

Genomic DNA from SM1 Genomic DNA from SM1 biofilmbiofilm was shown to have was shown to havea G+C content of 35.0 mol %.a G+C content of 35.0 mol %.

Transmission Electron Microscopy (TEM) indicatedTransmission Electron Microscopy (TEM) indicatedsmall small coccoidcoccoid archaealarchaeal cells embedded in highly cells embedded in highlycomplex cell appendages (comparable to complex cell appendages (comparable to hamihami).).

UltrastructuralUltrastructural TEM indicated that the TEM indicated that the hamihami had a had ahook and prickle structurehook and prickle structure

CLSM studies/SM1 CLSM studies/SM1 biofilmbiofilmoccurrence in other biotopesoccurrence in other biotopes

CLSM studies indicated a constant three-dimensionalCLSM studies indicated a constant three-dimensionalarrangement with individual cells 4 µm apart.arrangement with individual cells 4 µm apart.

Bacteria associated with Bacteria associated with archaealarchaeal biofilmbiofilm were distributed were distributedthroughout throughout biofilmbiofilm layers and arranged in small layers and arranged in small microcoloniesmicrocoloniesof 3 to 20 cells.of 3 to 20 cells.

Majority of cells had an active physiological stateMajority of cells had an active physiological state All cells showed strong hybridization signalsAll cells showed strong hybridization signals EuryarchaealEuryarchaeal SM1 SM1 biofilmbiofilm were shown to grow on were shown to grow on

polyethylene nets in different springs of the polyethylene nets in different springs of the SipenauerSipenauer Moor. Moor.

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DiscussionDiscussion

The results show that SM1 The results show that SM1 euryarchaeaeuryarchaeaare able to form a naturally occurringare able to form a naturally occurringmonospeciesmonospecies biofilmbiofilm..

This is the first time such a phenomenonThis is the first time such a phenomenonhas been detected.has been detected.

Proved that the in situ Proved that the in situ biofilmbiofilm trapping trappingmethod was effective.method was effective.

DiscussionDiscussion The formation of tiny The formation of tiny biofilmsbiofilms after only 10 after only 10

minutes suggest that the minutes suggest that the biofilmsbiofilms already alreadyexisted in the subsurface.existed in the subsurface.

The three dimensional arrangement of theThe three dimensional arrangement of theSM1 cells is likely due to 2 µm SM1 cells is likely due to 2 µm hamihami..

DiscussionDiscussion

Electron dense corona most likely due toElectron dense corona most likely due topolysaccharide capsule.polysaccharide capsule.

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What Next?What Next?

Can further analyze the relationship between the deltaCan further analyze the relationship between the deltaproteobacteriaproteobacteria and the SM1 and the SM1 euryarchaeoneuryarchaeon in the SM1 in the SM1biofilmbiofilm..

Further investigate whether SM1 Further investigate whether SM1 euryarchaeoneuryarchaeon have an have ananaerobic (sulfate reducing) metabolism.anaerobic (sulfate reducing) metabolism.

Investigate Investigate hamihami’’ss role in role in biofilmbiofilm formation. formation. Research into the Research into the existanceexistance of a polysaccharide capsule. of a polysaccharide capsule. Research into extent of SM1 Research into extent of SM1 biofilmsbiofilms in the subsurface. in the subsurface. Investigate SM1 Investigate SM1 biofilmsbiofilms’’ role in biogeochemical role in biogeochemical

activities and activities and geomicrobiologicalgeomicrobiological processes. processes.