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Natural Guided Genome Engineering Reveals Transcriptional Regulators Controlling Quorum-Sensing Signal Degradation.

El Sahili A, Kwasiborski A, Mothe N, Velours C, Legrand P, Moréra S, Faure D - PLoS ONE (2015)

Bottom Line: QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL).Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal.This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France.

ABSTRACT
Quorum-quenching (QQ) are natural or engineered processes disrupting the quorum-sensing (QS) signalling which controls virulence and persistence (e.g. biofilm) in numerous bacteria. QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL). Rhodococcus erythropolis known to efficiently degrade NAHL is proposed as a biocontrol agent and a reservoir of QQ-enzymes for biotechnology. In R. erythropolis, regulation of QQ-enzymes remains unclear. In this work, we performed genome engineering on R. erythropolis, which is recalcitrant to reverse genetics, in order to investigate regulation of QQ-enzymes at a molecular and structural level with the aim to improve the QQ activity. Deep-sequencing of the R. erythropolis enhanced variants allowed identification of a punctual mutation in a key-transcriptional factor QsdR (Quorum sensing degradation Regulation) which regulates the sole QQ-lactonase QsdA identified so far. Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal. This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated.

No MeSH data available.


Expression of the qsaRAB genes.RT-qPCR monitoring of the qsaRAB expression in R. erythropolis R138 wt and its mutants M7.1 and M7.3 grown in the presence of mannitol (AB-man) and OC8HSL (AB-OC8HSL) as a sole carbon source. Expressions were normalized using the recA gene as a reference gene. Experiments were done in triplicate.
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pone.0141718.g002: Expression of the qsaRAB genes.RT-qPCR monitoring of the qsaRAB expression in R. erythropolis R138 wt and its mutants M7.1 and M7.3 grown in the presence of mannitol (AB-man) and OC8HSL (AB-OC8HSL) as a sole carbon source. Expressions were normalized using the recA gene as a reference gene. Experiments were done in triplicate.

Mentions: R138 wild-type, the gene qsaR is divergently transcribed from two adjacent genes, that we named qsaA and qsaB, coding for an amidohydrolase (CDS819) and a transporter (CDS820) of the Major Facilitator Superfamily (MFS), respectively. In the wild-type strain R138 and its derivatives M7.1 and M7.3, the expression of genes qsaR, qsaA and qsaB was monitored by RT-qPCR in the presence of mannitol or OC8HSL as a sole carbon source (Fig 2). All the genes exhibited a higher transcription level in the evolved backgrounds as compared to that observed in the wild-type strain whatever the culture medium.


Natural Guided Genome Engineering Reveals Transcriptional Regulators Controlling Quorum-Sensing Signal Degradation.

El Sahili A, Kwasiborski A, Mothe N, Velours C, Legrand P, Moréra S, Faure D - PLoS ONE (2015)

Expression of the qsaRAB genes.RT-qPCR monitoring of the qsaRAB expression in R. erythropolis R138 wt and its mutants M7.1 and M7.3 grown in the presence of mannitol (AB-man) and OC8HSL (AB-OC8HSL) as a sole carbon source. Expressions were normalized using the recA gene as a reference gene. Experiments were done in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4640858&req=5

pone.0141718.g002: Expression of the qsaRAB genes.RT-qPCR monitoring of the qsaRAB expression in R. erythropolis R138 wt and its mutants M7.1 and M7.3 grown in the presence of mannitol (AB-man) and OC8HSL (AB-OC8HSL) as a sole carbon source. Expressions were normalized using the recA gene as a reference gene. Experiments were done in triplicate.
Mentions: R138 wild-type, the gene qsaR is divergently transcribed from two adjacent genes, that we named qsaA and qsaB, coding for an amidohydrolase (CDS819) and a transporter (CDS820) of the Major Facilitator Superfamily (MFS), respectively. In the wild-type strain R138 and its derivatives M7.1 and M7.3, the expression of genes qsaR, qsaA and qsaB was monitored by RT-qPCR in the presence of mannitol or OC8HSL as a sole carbon source (Fig 2). All the genes exhibited a higher transcription level in the evolved backgrounds as compared to that observed in the wild-type strain whatever the culture medium.

Bottom Line: QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL).Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal.This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France.

ABSTRACT
Quorum-quenching (QQ) are natural or engineered processes disrupting the quorum-sensing (QS) signalling which controls virulence and persistence (e.g. biofilm) in numerous bacteria. QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL). Rhodococcus erythropolis known to efficiently degrade NAHL is proposed as a biocontrol agent and a reservoir of QQ-enzymes for biotechnology. In R. erythropolis, regulation of QQ-enzymes remains unclear. In this work, we performed genome engineering on R. erythropolis, which is recalcitrant to reverse genetics, in order to investigate regulation of QQ-enzymes at a molecular and structural level with the aim to improve the QQ activity. Deep-sequencing of the R. erythropolis enhanced variants allowed identification of a punctual mutation in a key-transcriptional factor QsdR (Quorum sensing degradation Regulation) which regulates the sole QQ-lactonase QsdA identified so far. Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal. This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated.

No MeSH data available.