# Presentation: 463525

## Natural Gene Transfer to Develop Resistance to Metal Toxicity in Microbial Communities at the Oak Ridge FRC

**Jeffrey Fitts, Garry Crosson (****BNL Environmental Sciences****)**
**Daniel Van der Lelie, David Moreels, Safiyh Taghavi, Craig Garafola (****BNL Biology Department****)**

**Project Number EE-595-EEDA**

## FRC conditions present challenges to in-situ bioremediation strategies 

***Acidity***
***Heavy metals (Ni, Al...)***

**FRC conditions present challenges to in-situ bioremediation strategies **

- Will introduction of nickel resistance into indigenous microbial community have a positive effect on nitrate reducers and stimulate iron and sulfate reducers?

**Problematic conditions**

***Too high redox for stimulating sulfate and Fe reducers***
***Affects metal bioavailability, and thus, toxicity ***
***Inhibit nitrate reducers***

**Result**

## Project overview

**S3 ponds at ORNL**

_***Goal:***_*** Immobilize uranium in contaminated sediments via microbial reduction and precipitation***
_***Problem:***_*** Active uranium reducers are inhibited by co-contaminants in complex waste streams (e.g., heavy metals)***

_***Major project objectives***_
***Demonstrate application of natural gene transfer to improve community function under increased levels of toxic metal stress (******van der Lelie, BNL Biology Dept******.)***
***Demonstrate ability to enhance uranium immobilization in ORNL sediments by indigenous microorganisms that have adopted the toxic metal resistance marker*** ***(******Fitts, BNL Environmental Sci. Dept******.)***

## Project design schematic – presentation outline 

- FRC soils

- Model organisms

*Isolated from FRC fluidized bed reactor*

- _Nickel stress_
- 1. Community structure
- 2. Improved nitrate red.

- Total community

*Horizontal gene transfer (in vivo) – soil columns*

- Ni resistance

*Strain construction*
*(in vitro)*

- 1. S and Fe reduction
- 2. Uranium reduction and precipitation

*mechanisms*

**Notes:**
Today we present results of the effect of nickel on the indigenous community structure and function to establish a baseline for identifying bioaugmentation provided by introducing nickel resistant organisms into the system and assess HGT

## Construction of model organisms

***Mini Tn5 single hopper transposons: provide stability due to loss of transposase gene***

**Construction of model organisms**

*Ralstonia*
*Cupriavidus*
- Metal resistant gene

- Minimum required for Ni resistance: *nreB*

**Notes:**
Gene provides resistance to a number of metals including Ni, for which it has been shown that the this portion of the nre fragment is responsible for nickel resistance
Introduce nickel resistance by taking a metal resistant gene from cupriavidus metallurdurans and introducing it via conjugation
Metal resistance resistant gene will be introduced either as a plasmid (pMOL222) or genetic insertion (Tn5 mini hopper transposon)
It is hypothesized that the plasmid will be more efficient at HGT from the model organism to the community; and parallel column experiments with insertion and the plasmid will enable us to test this hypothesis.
***Stability tests in culture show plasmid is stable, but it might be more prone to being lost in vivo; Come back to this at the end w/ qPCR of community for nre;  

## nre gene provides Ni resistance to broad range of strains

*- proven concept (Dong et al., 1998; Taghavi et al., 2001)*
*Kanamycin selection marker *

***nre***** gene provides Ni resistance to broad range of strains**

**Notes:**
Demonstrated in Dr. van der Lelie’s lab that nre provides resistance to proteo and gram positive bacteria by introducing on the transposon shown below - insertion;
Kanamyacin provides independent selection for determining successful insertion – another way to look for HGT in the columns, for example if we don’t see nickel resistance increase, there may still be HGT;
***Problem with using kanamycin discovered in spread plating.

## Ni resistance introduced into nitrate reducers

***MIC values independent of plasmid vs. genomic insertion***

**Ni resistance introduced into nitrate reducers**

**Notes:**
Ni-sensitive wild type doesn’t grow in the presence of nickel - <0.5mM;
Two strains of psuedomonas that have a nickel resistant wild type; 
Higher resistance in aerobic vs. anaerobic;
In addition to observing increased nickel resistance we confirmed gene transfer on the genome level using PCR

## Successful gene transfer is confirmed on the genome level

- Both plasmid and genomic insertion confirmed for all strains

*Pseudomonas* DM-Y2 pMOL222

- n°2

- n°3

- n°7

- 1   2    3   4    5   6

- 1   2    3   4    5   6   *

- 1   2    3   4    5   6   *

_**BOX PCR**_
- 1: receptor P. DM-Y2
- 2: transconjugant
- 3: Donor E. coli CM2034 (in case of ncc-nre E. coli CM 2520)
_***nreB***__** PCR**_
- 4: receptor P. DM-Y2
- 5: transconjugant
- 6: Donor E. coli CM2034 (in case of ncc-nre E. coli CM 2520)
- * 1 kb ladder

*nre* in P.DMY2

- not *E. coli*

*nre* in E. coli

**Notes:**
Select for kanamycin, nickel and nutrient
Box PCR provides a fingerprint of an organism;
We use box pcr to determine that it is psuedomonas and not the original donor, in this case e. coli;
See for the 3 transconjugants on this gel that we have DM-Y2
We do PCR with nre specific probes to confirm that Pseudomonas has the gene fragment which is essential for nickel resistance;
Therefore confirms the results of the MIC tests that the transconjugants of an increased resistance relative to the wild type as a result of the successful introduction of the nre gene

## Plasmid transfer produces equally stable nre gene

- Both plasmid and genomic insertion exhibit equal stability in culture

- after 100 generations

**Notes:**
How well is nreB being maintained in the organism without nickel pressure; 
After 100 generations growing on non-selective/rich media results are species dependent; 
Show examples – shewanella it is in and then out
We chose to work with psuedomonas in column studies b/c of its high stability which will improve the odds of observing HGT, even though it doesn’t have the highest MIC;
Important to note that plasmid and insertation have similar stabilities.

## Ni resistance mechanisms

***Scanning Transmission X-ray Microscope imaging at the carbon K-edge***
***Washed cells exposed to Ni for 2 hrs***
***Rinsed cells are dried on microscope window***

**Ni resistance mechanisms**

-  Carbonates not observed
-  O K-edge will be sensitive to NiO formation

**2.5********m**

- Carbonate in organic matrix

- Pseudomonas DMY2::ncc-nre exposed to 2 mM NiCl2

- C=C

- carbonate

- Optical Density at 290eV

- Cluster image

- NSLS Beamline X1A

**Notes:**
Also have an effort to understand how 

## Column experiments

***Homogenization under atmospheric conditions***
***Inoculated columns with extracted indigenous community ***
***Anaerobic mineral growth media w/ C:N:P of 100:10:1 (ethanol carbon source)***
***Operate under anaerobic conditions***

**Column experiments**

## Pseudomonas DMY2 tested in column studies

**Pseudomonas DMY2 tested in column studies**

- Media +
- Formaldehyde

- Media +
- 1 mM NiCl2

- Media
- no Nickel added

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**Kill**

**2,6 - FRC community**

**3,7 - FRC community + Pseudomonas wild type**

**4,8 - FRC community + Pseudomonas pMol222**

**5,9 - FRC community + Pseudomonas::ncc-nre**

**Notes:**
Use quantitative PCR to compare nre levels in columns with plasmid vs. genomic insertion – greater levels of nre in the plasmid column (after normalization) will indicate that gene transfer has occurred. Single hopper will not allow transfer to occur. 

## Geochemical interrogation: S, Fe & U at time zero

**Geochemical interrogation: S, Fe & U at time zero**

- U oxidation state at M5 edge

- S speciation and redox state

- U-Fe correlation

- U distribution

**port B**

- Typical soil
- Organic matter

- Area 2 FRC soil

- inorganic sulfate

- sulfate

- reduced organic S species

- Area 2 FRC soil

- U6+
- standard

- U4+
- standard

- NSLS beamlines X27A & X15B

**Notes:**
Uranium well distributed in as delivered sediments; Not surprisingly U and Fe are correlated in sediments; U in as delivered sediments is oxidized; M5 also gives us a measure of 
Measurements of batch studies show some displacement of U, consistent with extraction results

## Geochemistry of columns after 65 days

- Ni distribution in Column 2

*No reduction of Uranium observed*

*Small increase in sulfide relative to kill*
*   **(oxidation during transfer may be problem)*

*Fe(III) oxides still dominate *

*Initial mobilization of Uranium*

*Nickel breakthrough observed but significant adsorption occurs*

- _Soil indicators by x-ray absorption spectroscopy _

- _Column effluent indicators_

**Notes:**
Is nickel breakthrough observed?

## Column experiment after 78 days – nitrate analyses

- After 78 days significant nitrate reduction in the viable columns
- Negative effect of nickel on nitrate reduction

- Below detection

## Column experiment after 78 days – sulfate analyses

-  No significant reduction in sulfate is observed

## Community analysis – Most Probable Number Counts

- MPN Counts for nitrate, sulfate and iron reducers

- MPN for nitrate, sulfate and iron reducers
-  Increase of nitrate reducing & iron reducing organisms
-  Decrease of sulfate reducing organisms
-  Nickel has a negative effect on sulfate and iron reducing 	organisms

**Notes:**
Don’t see sulfate reducers

## Relative number counts of nickel and kanamycin resistant bacteria
Enrichment of nickel resistant organisms
More nickel resistant organisms under pressure of nickel

- Relative number counts of nickel and kanamycin resistant bacteria
- Enrichment of nickel resistant organisms
- More nickel resistant organisms under pressure of nickel

- Start

- 44 days with nickel

- 44 days without nickel

## Community analysis - ongoing

***- TOPO TA cloning: 100 clones***
***Presently being sequenced; community composition will be determined at least on genus level***

**Community analysis - ongoing**

**Notes:**
TOPO TA  clonong;
Targeted probes for 16s rDNA and recovered the section for the entire community;
Sequence 16s rDNA fragment with TOPO TA cloning: TOPO TA is a plasmid that accepts 16s rDNA fragments b/c the polymerase enzymes have added and adenine to each end;  insert in E. coli using heat shock; plate and then pick clones; extract DNA from clones, sequence 

## Findings and Future directions

***Determine community composition of columns bioaugmented with engineered Pseudomonas DMY2***
***Quantitative PCR with nre targets to look for evidence of horizontal gene transfer in FRC column communities***
***Ni bioavailability measured with Ni biosensor, based on lux fusion (light production) with cnr (nickel resistance operon) in strain CH34***
***Explore ******Cupriavidus metallidurans***** *****strain CH34 which has natural resistance to a variety of metals***
***Extend findings to other systems, e.g. Hg (II) and Cr(VI) resistance***

## Acknowledgements

- _NSLS Measurements  _
- Paul Northrup (BNL Environmental Sci. Dep.)  – XAS & Microprobe
- Bjorg Larson, Sue Wirick (Stony Brook University) – STXM
- James Ablett (BNL NSLS) – Microprobe beamline X27A

- _Oak Ridge FRC_
- Dave Watson (ORNL)

- _BNL Biology Department_
**David Moreels**, Safiyh Taghavi, Craig Garafola

- _BNL Environmental Sciences Dept._
**Garry Crosson**