# Presentation: 222517

## Stanford University

**Engineering combinatorial bacteria for superior**
**metal and radionuclide bioremediation**** **

**A. C. MATIN**

## Nuclear weapons production

**Nuclear weapons production**

## Cr(VI) vs. Cr(III)

- Cr (VI) (chromate)

- Cr(III)

- Rapid cellular uptake                Not taken up

- Carcinogenic                             Non

- -

- carcinogenic

- Soluble  (can spread)                Insoluble

**Cr(VI) vs. Cr(III)**

- Bacteria can reduce Cr(VI); hence interest

- in bioremediation

**U(VI)**

**U(IV)**

## Several bacteria can reduce chromate

|  |  |  |  |
| --- | --- | --- | --- |
|  | Activity (nmol/mg protein/h) |  |  |
|  | Grown without chromate | Grown with 0.4 mM chromate | Assayed in presence of 10 mM CuSO4b. |
| P. putida KT2440 | 8.4 | 27.6 | 52.8 |
| P. putida MKI | 13.2 | 42.6 | 72.0 |
| P. syringae | 151.8 |   N.D.c. | N.D. |
| P. fluorescens | 114.6 | N.D. | N.D. |
| B. subtilis | 15.6 | 33.0 | 40.8 |
| V. harveyi | 132.0 | 264.0 | 1440.0 |
| E. coli AB117 | 49.8 | 32.4 | 156.0 |
| S. putrefaciens | 193.2 | N.D. | N.D. |
| D. radiodurans | 186.6 | N.D. | N.D. |
| S. aureus | 139.2 | N.D. | N.D. |
| Acinetobacter | 133.8 | N.D. | N.D. |

_**Several bacteria can reduce chromate**_
_**using soluble enzymes**_

*Enterobacter*
*cloacae: anaerobic*
*chromate respirer?*

**Keyhan, Ackerley, & Matin. 2003. **
**Batelle Symposium Proc. Paper *****E06***

## Effect of chromate toxicity

**Effect of chromate toxicity**

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

## Shi & Dalal, using ESR, showedthat many enzymes (e.g., lipoyl dehydrogenase, glutathione reductase, and cytochrome b5 reductase generate Cr(V) from Cr(VI), indicating one-electron reduction

## Cr(V) redox cycling

**Sulfate Transporter**

**Cr****VI**

**Cr****III**

**Cr****VI**

**Protein damage**
**Lipid peroxidation**
**DNA damage**

_**Cr(V) redox cycling**_

**Lipoyl dehydrogenase**
**Glutathione reductase**
**Cytochrome *****b******5***** reductase**

**NAD(P)H**
**NAD(P)**

**Chromate redox cycling**

## NAD(P)H ELECTRONS TRANSFERRED TO

- <date/time>

**NAD(P)H ELECTRONS TRANSFERRED TO**
**CHROMATE & H****2****O****2**** (********M; 1 min)**

| Enzyme | NAD(P)H
e- consumed | e- used in
chromate 
reduction | e- used in H2O2 formation |
| --- | --- | --- | --- |
| 
LpDH
 | 
80 | 
21 (26%) | 
59 (74%)
 |
| Single electron reducer |  |  |  |

**Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. **
**Environmenal Microbiology **_**6**_**: 851-860**

**Lipoyl dehydrogenase**

## In vivo oxidative stress detected using the

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

**In vivo oxidative stress detected using the**
**ROS-responsive dye H****2 ****DCFDA**

## Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol

**2**

**1**

**2**

**1**

**8**

**9**

**10**

**11**

**12**

**13**

**14**

**15**

**16**

**8**

**9**

**13**

**14**

**Unchallenged**

**Challenged non-adapted**

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

**Partial snap-shot of proteomics of chromate challenge**

## Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

| spot # | protein | gene | fold-change | known or suspected function |
| --- | --- | --- | --- | --- |
| 3 h samples |  |  |  |  |
| 1 | Flagellin | fliC | 11-fold down | Flagellar subunit |
| 2 | ADP-L-glycero-D-manno-heptose-6-epimerase | hldD | 4.1-fold down | Lipopolysaccharide biosynthesis |
| 5 h samples |  |  |  |  |
| 3 | Superoxide dismutase (ferric; chain B) | sodB | +++ | Decomposition of O2.- |
| 4 | Sulfate adenylyl transferase | cysN | 6.3-fold up | Cysteine biosynthesis |
| 5 | Cysteine synthase A | cysK | 4.1-fold up | Cysteine biosynthesis |
| 1 | Flagellin | fliC | --- | Flagellar subunit |
| 2 | ADP-L-glycero-D-manno-heptose-6-epimerase | hldD | 3.8-fold down | Lipopolysaccharide biosynthesis |
| 6 | Cytoplasmic ferritin | ftnA | 3.8-fold down | Iron storage |
| 7 | Putative formate acetyl transferase | yfiD | 3.3-fold down | unknown |

**Induction also of *****sfiA***** gene, indicative**
**of activation of the SOS response**
**Depletion of cellular glutathione and **
**Other free thiols**

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

## Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol

| Strain |  |
| --- | --- |
| wild type | 400 |
| Wild type | 400 |
| cysK* | 300 |
| cysN* | 400 |
| sodA | 450 |
| sodB* | 350 |
| katE | 300 |
| katG | 400 |
| gshA | 400 |
| gshB | 400 |
| yieF | 300 |
| fliC* | 400 |
| ompW* | 450 |
| ftnA* | 400 |
| sspA* | 400 |

**Chromate**
**MIC****50**** (****μM)**

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

**Chromate toxicity increases in mutants missing anti-oxidant defense genes**

## HYPOTHESIS

**Bacteria possess functional homologues of mammalian NQO1 enzyme (DT diaphorase)**
**                           **________________

**NQO1 is an obligatory two-electron **
**reducer of its substrates**
**A dimer of a bacterial homologue should be **
**capable of one-step reduction of Cr(VI)**
**to Cr(III), bypassing:**
**Cr(V) generation**
**Redox cycling**
**Will generate minimal possible ROS during **
**Cr(VI) reduction**

## Chromate reduction involving one-step

**Sulfate transporter**

**Cr****VI**

**Cr****III**

**Cr****VI**

**Stoichiometric and minimal ROS generation**

**4 electrons donated simultaneously – dimer of an obliatory 2-e****-**** tranferer**

**Chromate reduction involving one-step**
**conversion to Cr(III)  **

## 1. Classical biochemical methods: P. putida chromate reductase purification (Park,  Keyhan, Wielinga, Fendorf, & Matin  2000, AEM  66: 1788-1795)

***3. In-silico***** approaches**
*(Park et al., 2002, Remediation and beneficial use of *
*Contaminated sediments, p, 103-111)*
***4. Gene cloning; homo-***
***logues***
***5. Pure proteins using ***
***molecular techniques***

***2.***** Serendipity**

**NO  ASSIGNED  FUNCTION**

## NAD(P)H electrons transferred to

- <date/time>

**NAD(P)H electrons transferred to**
**Chromate & H****2****O****2**** by simultaneous e****-**** transfer**

| Enzyme | NAD(P)H
e- consumed | e- used in
chromate 
reduction | e- used in H2O2 formation |
| --- | --- | --- | --- |
| YieF | 62 | 46 (74%) | 16 (26%) |
| Simultaneous four electron transfer |  |  |  |

**Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. Environmenal Microbiology **_**6**_**: 851-860**

## Confirmation

** ****Rapid scan spectrophotometry to detect enzyme intermediates - ****Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. AEM **_**70**_**: 873-882**
**Electron Spin resonance studies**** -****Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. Environmenal Microbiology **_**6**_**: 851-860**
**                       **

## Three “safe” enzyme pure proteinscharacterized in detail

**ChrR of *****P. putida*****: semitight**
**NfsA of *****E. coli *****– semitight**
**Yief of *****E. coli***** – tight**
**YieF is the safest (generates least possible amount of ROS)**
**ChrR and NfsA permit some redox cycling but are much safer than one-electron reducers, generating only a fraction of ROS compared to the latter.**
**                        **

## In vivo oxidative stress detected using the

**Ackerley, Barak, Lynch, Curtin, and Matin. 2006. J. Bacteriol**
_**188**_** *****In press***

**In vivo oxidative stress detected using the**
**ROS-responsive dye H****2 DCFDA**

## Strategy to detoxify chromate reduction

**Sulfate transporter**

**Cr****VI**

**Cr****III**

**Cr****VI**

**Two e****-  ****reducers**

**Cr****III**

**One e****-**** reducers,**
**LpDH and others**

**Decreased ROS**

_**Strategy to detoxify chromate reduction**_

**Increase activity and affinity of two e****-**** reducers**

_**Enhanced activity**_

## Protection by over expression of 

_**Protection by over expression of **_
_**two-electron reducers**_

_**More chromate reduced per unit biomass**_

**Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. **
**Environmenal Microbiology **_**6**_**: 851-860**

## Slide 22

## Slide 23

## Cr(VI) reduction kinetics of YieF and the 

**Cr(VI) reduction kinetics of YieF and the **
**improved derivative Y6**

**Barak, Dodge, Francis a, nd Matin. In preparation**

**XANES Analysis showed quantative conversion to Cr(III)**
**Direct demonstration that Y6 also does not generate redox**
**Cyclinng**

**(30-fold)**

## Physiological role

** ****Broad substrate range: Quinones, ferricyanide, DCPIP, Mo (IV), and V(V).  ****Ackerley, Gonzalez, Park, Blake II, Keyhan, & Matin. 2004. AEM **_**70**_**: 873-882**
**General role is to protect against electrophiles like the ones mentioned above, with predilection for one-electron reduction and redox cycling **** - ****Gonzalez,  Ackerley, Lynch & Matin. 2005. Journal of Biological Chemistry **_**280:**_** 22590-595**
**                       **

## Recent results show that U(VI)reduction also subjects the bacterialcell to oxidative stress

**YieF & Other “safe” enzymes could reduce also U(VI) – activity detected only when overproduced**

## U(VI) reduction kinetics of YieF and the improved derivative Y6

**U(VI) reduction kinetics of YieF and the improved derivative Y6**

**Analysis showed quantitative conversion to U(IV)**
**Direct demonstration that Y6 also does not generate redox**
**cycling**
**Barak, Dodge, Francis a, nd Matin. In preparation**

**(12-fold)**

## Surprise application incancer prodrug chemotherapy

** ****Reductive prodrugs (e.g., CB 1954 and CNOB [discovered by us] become toxic only upon reduction**
**Y6 and other evolved enzymes are highly effective in this respect also**** -****Barak, Thorne, Contag, and Matin. 2006 Molecular**
**Cancer Therapeutics. **
**                       **

## Barak, Thorne, Contag, and Matin. 2006 Molecular

**Barak, Thorne, Contag, and Matin. 2006 Molecular**
**Cancer Therapeutics. **
**Stanford Patent S067**

## Slide 30

## Only one change, tyrosine128 to asparagine, responsible for all three improvementsApplication of a stochastic model predicted other beneficial changes; one enzyme has >830-fold improvementBarak, , Nov & Matin,, In preparation

## Work in progress

** ****Screening of shuffled libraries for other useful reductions, eg., Pu**** **
**Manipulate outer membrane porins to increase *****P. putida outer membrane permeability***
**Improve cytoplasmic**** ****for chromate transport**
**Combine activity with nitrate reduction**
** ****Batch and column studies of efficacy, survival and competitive ability in the presence of FRC material**
**                       **

## ACKNOWLEDGMENT

**C. H. Park                 B. Wielinga        R. Blake (II)**
**D. F. Ackerley           S. Fendorf           (Xavier)**
**C. Gonzalez**
**M. Keyhan**
**S. Lynch**
**Y. Barak**
**B. Salles**
**NABIR program of DOE **
**for supporting the research**