# Presentation: 458471

## Adaptation of  subsurface microbial communities to mercury

- Prof. Søren J. Sørensen (PI)
- Dept. of Microbiology, University of Copenhagen, Denmark
- Dr. Kroer’s group at  Dept. of Environmental Chemistry and Microbiology, National and Environmental Research Institute, Denmark
- Dr. Barkay’s group at Dept. of Biochemistry and Microbiology, Rutgers University, USA
- Prof. Smets group at Dept. of Civil & Environmental Engineering, University of Connecticut, USA

## Mercury Cycle in the Biosphere

**HS**

** ****-**

**H**

**g**

**0**

**H**

**g**

**2**

**+**

**3**

**(**

**C**

**H**

**3**

**)**

**2**

**H**

**g**

**H**

**g**

**0**

**H**

**g**

**0**

**+**

**H**

**g**

**(**

**p**

**)**

**,**

**3**

**H**

**g**

**2**

**+**

**H**

**g**

**0**

**3**

**C**

**H**

**HgCl**

**,**

**H**

**g**

**0**

**HgS**

**+**

- 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 soils

**Time (days)**

- μg **Hg/g soil**

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

## Contaminated subsurface soil

- de Lipthay, JR et al 2006 in prep

## Project tasks

- Isolation and characterization of hitherto uncultured bacteria of relevance for biotransformation of metals *(University of Copenhagen, NERI)*
- Horizontal gene transfer to “non-culturable” subsurface bacteria *(University of Copenhagen)*
- Significance of mobile genetic elements for microbial community adaptation to pollutant stress *(Rutgers University, University of Copenhagen)*
- Biostimulation of transformation rates in ground water and sediment *(University of Connecticut)*

## Soil samples

- A

- 0 m

- 0.5  m

- 1 m

- soil depth

- D

- C

- B

- Hinds Creek
- Floodplain

- Ish Creek Floodplain

- G

- F

- E

- Lower East Fork Poplar
- Creek Floodplain

| Soil | Site  | Depth  | Hg status | Total Hg | Bioavailable Hg |
| --- | --- | --- | --- | --- | --- |
|  |  | (inch) |  | (ug 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 plates

- The abundance of Hg resistant bacteria was higher in the contaminated soils.

- de Lipthay, JR et al 2006 in prep

## Adaptation Test

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

- 0μg Hg++/ml

## Adaptation to Hg++

- The surface soils were better adapted to Hg than the subsurface soil
- The contaminated soils were better adapted to Hg than noncontaminated

- de Lipthay, JR et al 2006 in prep

## Conclusions

- 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.

## Exogenic plasmid isolation

- Soil Slurry

- Recipient bacteria

- 	12 replicate isolations experiments from each soil with *E.coli* and *P.putida* as recipient bacteria

| Plasmid | Soil B | Soil C | Soil D | Soil E | Soil A |
| --- | --- | --- | --- | --- | --- |
| p1 | + | + | + | - | - |
| p2 | + | - | - | - | - |
| p2 | + | - | - | - | - |
| p4 | + | - | - | - | - |

## Exogenous plasmid isolation

-         p1      p2      p3       p4

- The four different plasmids were all beloning to the same Inc P1-beta group

- The plasmids and EcoRI Restriction cutting

| Plasmid | Recipient | Size | Transfer efficiency |
| --- | --- | --- | --- |
| p1 | E.coli | 57.3 Kb | 7.58 x 10-6 |
| p2 | P.putida | 33.6 Kb | 1.12 x 10-7 |
| p3 | E.coli | 68.1 Kb | 4.69 x 10-4 |
| p4 | E.coli | 36.0 Kb | 5.59 x 10-6 |

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

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

The merR gene encodes a Hg responsive regulator. 

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

**Gram-negative mercury resistance**

- The *merA* gene encodes a mercuric reductase that reduces Hg++ to volatile Hg(0).
- MerP and MerT are involved in the transportation of Hg++ to MerA in the cytosol.
- The *merR* gene encodes a Hg responsive regulator.

## Prokaryotic mercuric reductase protein diversity

## 16S rDNA clone library

## merA PCR

- 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.

*merA* PCR

-  Initially, *merA* genes were only found in control soils from the contaminated site.
-  Following soil Hg amendment, *merA* genes were found in all soils - indicating the adaptive potential of the subsurface soils.

- de Lipthay, JR et al 2006 in prep

## Slide 17

##  Cultivation of Hg++ resistant subsurface bacteria

- Microcultivation approach that simulates the natural growth conditions
- See our poster tonight!
- Use dilute media as proposed by Janssen et al. (AEM 2002) and long-term incubation (6-12 weeks)

## Cultivation a’ la Janssen

- Oregaard, G et al 2006 in prep

## Cultivation of Hg tolerant bacteria

- Oregaard, G et al 2006 in prep

## Direct detection of HGT

- Transconjugant cell

## Flow cytometry

- 488nm Laser

- Detectors

- FL1

- Green

- Yellow

- Size and surface

- FL2

- FSC

- SSC

- Red

- FL3

## Direct detection of HGT

- Rhizosphere 			12-38%	100-1000

- Cultivation based detection underestimate
-  the frequency of HGT

- Control (no donor)

- Transconjugants

-       Donors

- T/D

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

## Sorting transconjugant bacteria for molecular analysis

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

## Conclusions

- We have isolated several Hg++ resistance plasmid from subsurface bacteria.
- We can culture hitherto unculturable Hg++ tolerant subsurface bacteria.
- We have a new method for detecting plasmid transfer between subsurface bacteria without cultivation.
- We have developed DNA-microarray for characterization of plasmids.

- In the last year we will characterize and tag the isolated plasmids. Then we will study the transfer at *in situ* conditions and evaluate the possibility to use HGT as a mean to stimulate the heavy metal transformation.