molecular microbial ecology group department of microbiology 1 adaptation of subsurface microbial...

Molecular Microbial Ecology GroupMolecular Microbial Ecology Group Department of MicrobiologyDepartment of Microbiology11

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Adaptation of Adaptation of subsurface microbial communities subsurface microbial communities

to mercuryto mercury

Prof. Søren J. Sørensen (PI)Prof. Søren J. Sørensen (PI)Dept. of Microbiology, University of Copenhagen, DenmarkDept. of Microbiology, University of Copenhagen, Denmark

Dr. Kroer’s group at Dept. of Environmental Chemistry and Microbiology, National and Environmental Research Dr. Kroer’s group at Dept. of Environmental Chemistry and Microbiology, National and Environmental Research Institute, DenmarkInstitute, Denmark

Dr. Barkay’s group at Dept. of Biochemistry and Microbiology, Rutgers University, USADr. Barkay’s group at Dept. of Biochemistry and Microbiology, Rutgers University, USA

Prof. Smets group at Dept. of Civil & Environmental Engineering, University of Connecticut, USAProf. Smets group at Dept. of Civil & Environmental Engineering, University of Connecticut, USA




Mercury Cycle in the BiosphereMercury Cycle in the Biosphere

HS -

H g0

H g2+

3C H H g+

(C H 3)

2H g

H g0

H g0 + (C H 3

)2H g

3C H H g+

H g (p)H g0,

3C H H g+

H g2+

H g0

(C H 3)

2H g

3C H HgClH g (p)H g0, ,

H g0

H g2

H g0+ O 3

HgS +

2 +Hg (C H 3)

2H g

3C H H g+

Knowledge on the response and adaptation of soil bacterial communities to heavy metals is of high relevance as these contaminants are widely distributed in terrestrial environments. Heavy metals as mercury (Hg) arise from e.g. spread of fertilisers, and burning of fossil fuels - and naturally by weathering of rocks.

Microorganisms have the ability to transform several heavy metals and remove them from the aqueous phase thereby significantly reducing the risks associated with these pollutants. Bioremediation therefore seems a promising tool in the clean-up of contaminated environments.




Mercury contaminated soilsMercury contaminated soils

Time (days)Time (days)

µg µg

Hg

/g s

oil

Hg

/g s

oil

00

22

44

66

88

1010

00 22 44 66 88 1010 1212 1414 1616

HgHg++++ resistant CFU (%) resistant CFU (%)

00

11

22

33

44

00 22 44 66 88 1010 1212 1414 1616

Total HgTotal Hg++++

00

0,010,01

0,020,02

0,030,03

0,040,04

0,050,05

0,060,06

00 22 44 66 88 1010 1212 1414 1616

Bioavailable HgBioavailable Hg++++

1515

2020

2525

3030

3535

00 22 44 66 88 1010 1212 1414 1616

BiodiversityBiodiversity

no. bands on DGGEno. bands on DGGE

Rasmussen, L.D et al 2000 Soil Biol. Biochem 32, p639-646Rasmussen, L.D et al 2000 Soil Biol. Biochem 32, p639-646




Can we introduce Hg-resistance plasmids in natural bacterial populations, and thereby speed up adaptation and stimulate

microbial activities in contaminated subsurface soils?

de Lipthay, JR et al 2006 in prepde Lipthay, JR et al 2006 in prep

Donor, recipient and transconjugant numbersof microcosm A

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

0 7 14 21 28 35

Day

CFUs g-1 soil

A1 - donor

A1 - recipient

A1 - transconjugant

A2 - donor

A2 - recipient

A2 - transconjugant

detection limit

Contaminated subsurface soilContaminated subsurface soil




1) Isolation and characterization of hitherto uncultured bacteria of relevance for biotransformation of metals (University of Copenhagen, NERI)

2) Horizontal gene transfer to “non-culturable” subsurface bacteria (University of Copenhagen)

3) Significance of mobile genetic elements for microbial community adaptation to pollutant stress (Rutgers University, University of Copenhagen)

4) Biostimulation of transformation rates in ground water and sediment (University of Connecticut)

Project tasksProject tasks




Soil samplesSoil samples

A 0 m

0.5 m

1 m

soil

dept

h

D

C

B

Hinds CreekHinds CreekFloodplainFloodplain

Ish Creek Ish Creek Floodplain Floodplain

G

F

E

Lower East Fork PoplarLower East Fork PoplarCreek Floodplain Creek Floodplain

SoilSoil Site Site Depth Depth Hg statusHg status Total HgTotal Hg Bioavailable HgBioavailable Hg

(inch)(inch) (ug pr g soil)(ug pr g soil) (ng pr g soil)(ng pr g soil)

Soil B Poplar Creek 0-2" Contaminated 12.5 +/- 1.4 < d.l.

Soil C Poplar Creek 18-22" Contaminated 7.6 +/- 1.0 0.83 +/- 0.43

Soil E Ish Creek 0-2" Reference 0.2 +/- 0.05 < d.l.

Soil F Ish Creek 18-22" Reference 0.06 +/- 0.01 < d.l.




CFUs on mercury amended R2A platesCFUs on mercury amended R2A plates

The abundance of Hg resistant bacteria was higher

in the contaminated soils.

0,01

0,1

1

10

100

soil B soil C soil E soil F

% CFU on R2A with 5 µg/ml Hg(++) of total CFU





Adaptation TestAdaptation Test

EcoPlates containing 3 x 31 different sole EcoPlates containing 3 x 31 different sole carbon sources and a carbon sources and a tetrazolium redox dyetetrazolium redox dye

0µg Hg0µg Hg++++/ml/ml

1µg Hg1µg Hg++++/ml/ml




Adaptation to HgAdaptation to Hg++++

The surface soils were better The surface soils were better adapted to Hg than the adapted to Hg than the subsurface soilsubsurface soil

The contaminated soils were The contaminated soils were better adapted to Hg than better adapted to Hg than noncontaminatednoncontaminated

0

20

40

60

80

100

soil B soil C soil E soil F

% of carbon sources transformed





The mercury tolerance of the bacterial communities was higher in the contaminated soils than in the control soils despite very low bioavailable mercury concentrations in both types of soils. This indicates that an exposed soil will maintain its ability to tolerate mercury - even when the exposure is low.

The high adaptive potential of subsurface microbial communities suggest - similar to surface soils – that transfer of the mer operon by horizontal gene exchange may play a role for community adaptation to the applied mercury stress.

ConclusionsConclusions




Exogenic plasmid isolationExogenic plasmid isolationSoil SlurrySoil SlurryRecipient bacteriaRecipient bacteria

12 replicate isolations 12 replicate isolations experiments from each soil experiments from each soil with with E.coliE.coli and and P.putidaP.putida as as

recipient bacteriarecipient bacteria

Plasmid Soil B Soil C Soil D Soil ESoil E Soil ASoil A

p1 + + + -- --

p2 + - - -- --

p2 + - - -- --

p4 + - - -- --




Exogenous plasmid isolationExogenous plasmid isolation

p1 p2 p3 p4

The four different plasmids The four different plasmids were all beloning to the were all beloning to the

same Inc P1-beta groupsame Inc P1-beta group

The plasmids and EcoRI Restriction cuttingThe plasmids and EcoRI Restriction cutting

PlasmidPlasmid RecipientRecipient SizeSize Transfer Transfer efficiencyefficiency

p1 E.coli 57.3 Kb57.3 Kb 7.58 x 10-6

p2 P.putida 33.6 Kb33.6 Kb 1.12 x 10-7

p3 E.coli 68.1 Kb68.1 Kb 4.69 x 10-4

p4 E.coli 36.0 Kb36.0 Kb 5.59 x 10-6




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are needed to see this picture.

Barkay et al. FEMS microbiology Reviews 2003 27:355-384

Gram-negative mercury resistance

The The merAmerA gene encodes a gene encodes a mercuric reductase that mercuric reductase that reduces Hgreduces Hg++++ to volatile Hg(0). to volatile Hg(0).

MerP and MerT are involved in MerP and MerT are involved in the transportation of Hgthe transportation of Hg++++ to to MerA in the cytosol.MerA in the cytosol.

The The merRmerR gene encodes a Hg gene encodes a Hg responsive regulator.responsive regulator.




Prokaryotic mercuric reductase protein diversityProkaryotic mercuric reductase protein diversity

Actinobacteria

Proteobacteria

Archaea

Firmicutes




16S rDNA clone library16S rDNA clone library

AlphaAlphaBetaBeta

FirmicutesFirmicutesActinobacteriaActinobacteriaAcidobacteriaAcidobacteria

GemmatimonadetesGemmatimonadetes

GammaGammaNitrospiraNitrospira

MiscellaneousMiscellaneous

VerrucomicrobiaVerrucomicrobia

PlanctomycesPlanctomyces

DeltaDelta

BacteroidetesBacteroidetes




Soil B Soil C Soil D Soil E Soil F

Control + + + + + - + + + - - - - - -Hg amended + + + + + - + + + + + - + + +

+: merA present; -: merA abscentThe table indicates in how many of three replicate soil samples merA could be detected.

Figure show representative gel of one replicate

Lane 1: positive control (plasmid pHG103). Lane 15: negative control (H2O). Lanes 2, 9 & 16: 100 bp ladder.Lanes 4+10: soil B; lanes 5+11: soil C; lanes 6+12: soil D; lanes 7+13: soil E; lanes 8+14: soil F.Lanes 4-8 show data from the Hg amended soils, while lanes 10-14 show data from the control soils.

merAmerA PCR PCR

Initially, Initially, merAmerA genes were genes were only found in control soils from only found in control soils from the contaminated site.the contaminated site.

Following soil Hg Following soil Hg amendment, amendment, merAmerA genes were genes were found in all soils - indicating the found in all soils - indicating the adaptive potential of the adaptive potential of the subsurface soils.subsurface soils.




Der kræves QuickTime™ og et Photo - JPEG-komprimeringsværktøj,for at man kan se dette billede. Cultivation of HgCultivation of Hg++++ resistant resistant subsurface bacteriasubsurface bacteria

Microcultivation approach Microcultivation approach that simulates the natural that simulates the natural growth conditionsgrowth conditions See our poster tonight!See our poster tonight!

Use dilute media as Use dilute media as proposed by Janssen et proposed by Janssen et al. al. (AEM 2002)(AEM 2002) and long-term and long-term incubation (6-12 weeks)incubation (6-12 weeks)




Cultivation a’ la JanssenCultivation a’ la Janssen

1,0E+04

5,0E+06

1,0E+07

1,5E+07

2,0E+07

2,5E+07

3,0E+07

3,5E+07

7 14 21 29 35Day

CFU / g dry soil

B VL60Xyl B TSA C VL60Xyl C TSA

Oregaard, G et al 2006 in prepOregaard, G et al 2006 in prep




Cultivation of Hg tolerant bacteriaCultivation of Hg tolerant bacteria

1,0E+04

2,1E+05

4,1E+05

6,1E+05

8,1E+05

1,0E+06

1,2E+06

1,4E+06

1,6E+06

1,8E+06

2,0E+06

2,2E+06

2,4E+06

7 14 21 29 35Day

CFU / g dry soil

B VL60Xyl Hg B TSA Hg C VL60Xyl Hg C TSA Hg

Oregaard, G et al 2006 in prepOregaard, G et al 2006 in prep




Donor cellDonor cell

Recipient cellRecipient cell

Transconjugant cellTransconjugant cell

Direct detection of HGTDirect detection of HGT




Flow cytometryFlow cytometry

488nm Laser488nm Laser

DetectorsDetectors

FL1FL1 GreenGreen

YellowYellow

Size and surfaceSize and surface

FL2FL2

FSCFSC

SSCSSC

RedRedFL3FL3




Direct detection of HGTDirect detection of HGT

Rhizosphere 12-38% 100-1000

Cultivation based detection underestimateCultivation based detection underestimate the frequency of HGTthe frequency of HGT

Control (no donor)Control (no donor) TransconjugantsTransconjugants DonorsDonors

T/DT/D

Sørensen, SJ., et al 2005 Nature Reviews Microbiology 3 (9): p700-710Sørensen, SJ., et al 2005 Nature Reviews Microbiology 3 (9): p700-710



Der kræves QuickTime™ og et Photo - JPEG-komprimeringsværktøj,for at man kan se dette billede.Sorting transconjugant bacteria for Sorting transconjugant bacteria for molecular analysismolecular analysis

DGGE analysis of 16S rDNA clones DGGE analysis of 16S rDNA clones from sorted transconjugant cellsfrom sorted transconjugant cells

488nm Laser488nm Laser

DetectorsDetectors

FL1FL1 GreenGreen

Size and surfaceSize and surfaceFSCFSC

SSCSSC

Sørensen, SJ., et al 2005 Nature Reviews Microbiology 3 (9): p700-710Sørensen, SJ., et al 2005 Nature Reviews Microbiology 3 (9): p700-710




ConclusionsConclusions We have isolated several HgWe have isolated several Hg++++ resistance plasmid resistance plasmid

from subsurface bacteria.from subsurface bacteria. We can culture hitherto unculturable HgWe can culture hitherto unculturable Hg++++ tolerant tolerant

subsurface bacteria.subsurface bacteria. We have a new method for detecting plasmid transfer We have a new method for detecting plasmid transfer

between subsurface bacteria without cultivation.between subsurface bacteria without cultivation. We have developed DNA-microarray for We have developed DNA-microarray for

characterization of plasmids.characterization of plasmids.

In the last year we will characterize and tag the In the last year we will characterize and tag the isolated plasmids. Then we will study the isolated plasmids. Then we will study the

transfer at transfer at in situin situ conditions and evaluate the conditions and evaluate the possibility to use HGT as a mean to stimulate possibility to use HGT as a mean to stimulate

the heavy metal transformation. the heavy metal transformation.

molecular microbial ecology group department of microbiology 1 adaptation of subsurface microbial...

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