Team:Paris Saclay/Project

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[[Team:Paris_Saclay/Project|Overview]]
[[Team:Paris_Saclay/Project|Overview]]
[[Team:Paris_Saclay/Modeling|Modeling]]
[[Team:Paris_Saclay/Modeling|Modeling]]
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[[Team:Paris_Saclay/PS-PCR|PS-PCR]]
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[[Team:Paris_Saclay/Achievements|Achievements]]
{{Team:Paris_Saclay/incl_fin_menu_navigation}}
{{Team:Paris_Saclay/incl_fin_menu_navigation}}
{{Team:Paris_Saclay/incl_contenu}}
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= '''Detection and degradation of PCB system in E.coli''' =
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= '''Detection and degradation of PCB system in ''Escherichia coli''''' =
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Since the second half of the XXth century scientists are fully aware of the fact that some
+
Since the second half of the 20th century, scientists are fully aware that some bacterial species living in media with high concentration of PCBs are able to degrade PCBs into pyruvate and acetyl-CoA which are then easily metabolized by these organisms.
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species of bacteria living in mediums with high concentrations of PCB are able to degrade the
+
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PCB in pyruvate and acetyl CoA easily metabolized by these organisms.
+
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These species structure biofilms were there are regions with variables concentration of
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These bacterial species structure in biofilm with regions that have variable concentrations of oxygen, high at the surface and decreasing with depth. Bacteria living in this habitat have, in most cases, different degradation pathways, which are aerobic or anaerobic depending on their spatial disposition in the biofilm.
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oxygen, decreasing with depth to surface. The bacteria living in this habitat have in most of
+
-
case different degradation pathways namely aerobic or anaerobic depending on the spatial
+
-
disposition in the biofilm.
+
-
Bacteria in aerobic conditions use an aerobic degradation pathway, the PCB oxidative
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Bacteria in aerobic environment use PCB oxidative degradation pathways; those in anaerobic condition degrade PCBs via reductive dechlorination pathways. None of the bacteria seems to use both pathways.
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degradation, and these in anaerobic condition the PCB reductive dechlorination; no one can
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use both pathways so as to degrade the PCB.
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The reductive dechlorination can reduce the number of chlorines in high chlorinated PCB
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The reductive dechlorination reduces the number of chlorines of high chlorinated PCBs. The dechlorinated PCBs can be further degraded by an oxidative degradation which is efficient only with low chlorinated PCBs. That’s may explain why these different species coexist in biofilms.
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making them assimilable by the oxidative degradation, only efficient with low chlorinated
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PCB. That’s the reason why these different species coexist in the biofilms.
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'''So our goal in this project is the creation of an organism able to first detect PCB and after
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'''Our goal in this project is to desing an organism able to i) detect PCB and then ii)
'''employ a sequential degradation of the PCB using both combined pathways.'''
'''employ a sequential degradation of the PCB using both combined pathways.'''
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'''For our experiences we used bacteria present in nature that are able to detect and degrade the'''
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'''For our experiences, we used bacteria present in nature that are able to detect and degrade the'''
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'''PCB namely ''Burkholderia xenovorans, Pseudomonas pseudoalcaligenes KF 707,
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'''PCBs, namely [http://en.wikipedia.org/wiki/Burkholderia_xenovorans ''Burkholderia xenovorans''], [http://www.ncbi.nlm.nih.gov/pubmed/22843571''Pseudomonas pseudoalcaligenes'' KF 707] and
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'''Rhodococcus jostii RHA1''.''''''
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[http://en.wikipedia.org/wiki/Rhodococcus'''''Rhodococcus jostii'' RHA1].'''''
[[File:Psnotenough.png|center|200px]]
[[File:Psnotenough.png|center|200px]]
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==Detection and report of the PCB==
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==Construction of a system to detect the presence of PCBs==
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''Pseudomonas pseudoalcaligenes'' expresses enzymes that are responsible for an oxidative degradation of PCBs. The system is regulated two proteins, BphR2 and BphR1 coded by the ''bphR2'' and ''bphR1'' genes, respectively. PCBs induce a BphR2 conformational change to trigger BphR2 transcriptional activity leading to expression of the PCB oxidative degradation pathway.
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In nature these bacteria have a system for the regulation of the oxidative degradation of PCB.
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[[File:PsR2degradation.jpg]]
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This one is based on two regulatory proteins namely Bphr2 and Bphr1 coded respectively by
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the genes of the same name bphr2 and bphr1.
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 +
BphR2 also induces the expression of ''bphR1''.
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Bphr2 is able to detect PCB that induces a modification of the protein conformation activating
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[[File:PsR2surR1.jpg]]
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the beginning of the gene cluster coding for the enzymes doing the oxidative degradation but
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also the gene coding for the Bphr1 protein.
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 +
For the project, we will use the ''bphR2'' gene and the ''bphR1'' promoter. We will place the ''bphR2'' coding sequence under a constitutive promoter. We will also construct a transcriptional fusion between the ''bphR1'' promoter with the ''lacZ'' gene coding for the β-galactosidase enzyme. The amount of β-galactosidase can be easily monitored by a chemical reaction using Xgal. With this system, the β-galactosidase activity will dependent on the ''bphR1'' promoter expression. Since the ''bphR1'' promoter is controlled by the PCB-activated BphR2, the β-galactosidase activity will correlate with the presence of PCBs.
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The Bphr1 protein can detect the HO-PCB a metabolite derived from PCB, a product of the
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[[File:PsavecPCB.jpg|800px]]
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beginning of oxidation reactions. In presence of OH-PCB it induces his own transcription and
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also the following genes from the cluster that will completely degrade the PCB.
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For our construct we will pick out the bphr2 gene and the promoter of the bphr1 gene induced
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by PCB-Bphr2 from our species. We will combine the bphr2 coding sequence with a
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constitutive promoter that makes up the detection system and finally we will combine the
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Bphr1 promoter with the lacZ gene coding for the β-galactosidase enzyme so as to do a
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chemical dosing with Xgal and report the signal.
+
 +
==Combination of the aerobic and anaerobic PCB degradation pathways==
 +
To perform an efficient PCB degradation, two processes should be sequentially combined, the PCB reductive dechlorination followed by a PCB oxidative degradation pathway. Our goal is to engineer a bacterium expressing alternatively both pathways according to growth conditions, with first the reductive dechlorination in anaerobiosis followed by a PCB oxidative degradation in aerobiosis.
-
[[File:Psdegradationexplication.jpg|center]]
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The bacterium ''E. coli'' has an aerobic and an anaerobic metabolism that will be used to combine of the two PCB degradation pathways. The switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster. For the project, we use two promoters, PnrdH and PnifR which are repressed and activated by FNR, respectively
 +
The reductive dechlorination pathway is not well characterized; only one enzyme, a dehalogenase, is mentioned as contributing to this pathway. We propose to clone the corresponding gene in ''E. coli'' under the control of the PnifR promoter. As a result, the reductive dechlorination enzyme should be expressed in anaerobiosis to perform the first PCB degradation step. The second step involves an oxidative degradation and is performed in aerobiosis. We propose to clone the operon PCB oxidative degradation under the PrndH promoter which is derepressed in anaerobiosis. This configuration should optimize the PCB degradation according to the environmental conditions.
 +
[[File:Psbigschema.jpg|800px]]
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 +
==References==
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Kensuke Furukawa, Hikaru Suenaga and Masatoshi Goto
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 +
Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution
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JOUNAL OF BACTERIOLOGY, 2004
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 +
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Kazunari Taira, Jun Hirose, Shinsaku Hayashida, and Kensuke Furukawa
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 +
Analysis of bph Operon from the Polychlorynated Biphenyl-degrading Strain of ''Pseudomonas pseudoalcaligenes'' KF707
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THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1992
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Kensuke Furukawa and Hidehiko Fujihara
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Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features
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 +
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2008
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Jim A. Field, Reyes Sierra-Alvarez
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Microbial transformation and degradation of polychlorinated biphenyls
 +
 +
Environmental Pollution, 2008
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 +
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Lorenz Adrian, Helmut Görisch
 +
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Microbial transformation of chlorinated benzenes under anaerobic conditions
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Research in Microbiology, 2002
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 +
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Dean. A Tolla and Michael A. Savageau
 +
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Regulation of Aerobic-to-Anaerobic Transitions by the FNR Cycle in Escherichia coli
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J. Mol. Biol. (2010)
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==Combination of the aerobic and anaerobic PCB degradation pathways==
 
 +
Hidehiko Fujihara, Hideyuki Yoshida, Tetsuya Matsunaga, Masatoshi Goto, and Kensuke Furukawa
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The bacteria E.coli has an aerobic and an anaerobic metabolism that’s why we used it for the
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Cross-Regulation of Biphenyl- and Salicylate-Catabolic Genes by Two Regulatory Systems in Pseudomonas pseudoalcaligenes KF707
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combination of the two degradation pathways. The regulation between pathways in these two
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conditions is normally made by regulatory proteins like FNR.
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The FNR protein modifies its conformation in presence of oxygen having an activator or an
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inhibitor function.
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JOURNAL OF BACTERIOLOGY, July 2006
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The reductive dechlorination pathway is not well characterized only an enzyme, a
 
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dehalogenase, is mentioned as contributing to this pathway. In these anaerobic conditions the
 
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chlorine takes the place of the oxygen as the electron acceptor.
 
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That’s why we have chosen an activator FNR in presence of oxygen in order to activate the
 
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oxidative degradation.
 
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[[File:Psdegradationexplication2.jpg|center|800px]]
 
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Article written by Eric and Nadia
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 21:50, 4 October 2013

Project What are PCBs ? Overview Modeling PS-PCR Achievements

Contents

Detection and degradation of PCB system in Escherichia coli

Since the second half of the 20th century, scientists are fully aware that some bacterial species living in media with high concentration of PCBs are able to degrade PCBs into pyruvate and acetyl-CoA which are then easily metabolized by these organisms.


These bacterial species structure in biofilm with regions that have variable concentrations of oxygen, high at the surface and decreasing with depth. Bacteria living in this habitat have, in most cases, different degradation pathways, which are aerobic or anaerobic depending on their spatial disposition in the biofilm.


Bacteria in aerobic environment use PCB oxidative degradation pathways; those in anaerobic condition degrade PCBs via reductive dechlorination pathways. None of the bacteria seems to use both pathways.


The reductive dechlorination reduces the number of chlorines of high chlorinated PCBs. The dechlorinated PCBs can be further degraded by an oxidative degradation which is efficient only with low chlorinated PCBs. That’s may explain why these different species coexist in biofilms.


Our goal in this project is to desing an organism able to i) detect PCB and then ii) employ a sequential degradation of the PCB using both combined pathways. For our experiences, we used bacteria present in nature that are able to detect and degrade the PCBs, namely Burkholderia xenovorans, Pseudomonas pseudoalcaligenes KF 707 and Rhodococcus jostii RHA1.

Psnotenough.png


Construction of a system to detect the presence of PCBs

Pseudomonas pseudoalcaligenes expresses enzymes that are responsible for an oxidative degradation of PCBs. The system is regulated two proteins, BphR2 and BphR1 coded by the bphR2 and bphR1 genes, respectively. PCBs induce a BphR2 conformational change to trigger BphR2 transcriptional activity leading to expression of the PCB oxidative degradation pathway.

PsR2degradation.jpg

BphR2 also induces the expression of bphR1.

PsR2surR1.jpg

For the project, we will use the bphR2 gene and the bphR1 promoter. We will place the bphR2 coding sequence under a constitutive promoter. We will also construct a transcriptional fusion between the bphR1 promoter with the lacZ gene coding for the β-galactosidase enzyme. The amount of β-galactosidase can be easily monitored by a chemical reaction using Xgal. With this system, the β-galactosidase activity will dependent on the bphR1 promoter expression. Since the bphR1 promoter is controlled by the PCB-activated BphR2, the β-galactosidase activity will correlate with the presence of PCBs.

PsavecPCB.jpg

Combination of the aerobic and anaerobic PCB degradation pathways

To perform an efficient PCB degradation, two processes should be sequentially combined, the PCB reductive dechlorination followed by a PCB oxidative degradation pathway. Our goal is to engineer a bacterium expressing alternatively both pathways according to growth conditions, with first the reductive dechlorination in anaerobiosis followed by a PCB oxidative degradation in aerobiosis.

The bacterium E. coli has an aerobic and an anaerobic metabolism that will be used to combine of the two PCB degradation pathways. The switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster. For the project, we use two promoters, PnrdH and PnifR which are repressed and activated by FNR, respectively

The reductive dechlorination pathway is not well characterized; only one enzyme, a dehalogenase, is mentioned as contributing to this pathway. We propose to clone the corresponding gene in E. coli under the control of the PnifR promoter. As a result, the reductive dechlorination enzyme should be expressed in anaerobiosis to perform the first PCB degradation step. The second step involves an oxidative degradation and is performed in aerobiosis. We propose to clone the operon PCB oxidative degradation under the PrndH promoter which is derepressed in anaerobiosis. This configuration should optimize the PCB degradation according to the environmental conditions.

Psbigschema.jpg

References

Kensuke Furukawa, Hikaru Suenaga and Masatoshi Goto

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

JOUNAL OF BACTERIOLOGY, 2004


Kazunari Taira, Jun Hirose, Shinsaku Hayashida, and Kensuke Furukawa

Analysis of bph Operon from the Polychlorynated Biphenyl-degrading Strain of Pseudomonas pseudoalcaligenes KF707

THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1992


Kensuke Furukawa and Hidehiko Fujihara

Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features

JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2008


Jim A. Field, Reyes Sierra-Alvarez

Microbial transformation and degradation of polychlorinated biphenyls

Environmental Pollution, 2008


Lorenz Adrian, Helmut Görisch

Microbial transformation of chlorinated benzenes under anaerobic conditions

Research in Microbiology, 2002


Dean. A Tolla and Michael A. Savageau

Regulation of Aerobic-to-Anaerobic Transitions by the FNR Cycle in Escherichia coli

J. Mol. Biol. (2010)


Hidehiko Fujihara, Hideyuki Yoshida, Tetsuya Matsunaga, Masatoshi Goto, and Kensuke Furukawa

Cross-Regulation of Biphenyl- and Salicylate-Catabolic Genes by Two Regulatory Systems in Pseudomonas pseudoalcaligenes KF707

JOURNAL OF BACTERIOLOGY, July 2006




Article written by Eric and Nadia

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