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Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

Singh PK, Ramachandran G, Ramos-Ruiz R, Peiró-Pastor R, Abia D, Wu LJ, Meijer WJ - PLoS Genet. (2013)

Bottom Line: Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes.Ultimately, this peptide dictates the timing of conjugation.The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.

ABSTRACT
Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

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Model of regulatory circuitry of pLS20 conjugation genes.A. Repressed state due to RcoLS20. Gene rcoLS20 (red arrow, rco) encoding the master repressor of conjugation genes RcoLS20 is divergently transcribed from the putative conjugation operon encompassing genes 28 to 74 (light blue arrows). RcoLS20 inhibits expression of the conjugation genes by repressing a promoter, Pc, located upstream of gene 28, the first gene of the putative conjugation operon (our unpublished results). B. Activation of conjugation by RapLS20 anti-repressor. Gene rapLS20 (green arrow, rap) encodes the anti-repressor of RcoLS20 leading to de-repression of the conjugation genes. C. Repressed state due to inactivation of RapLS20 by signaling peptide Phr*LS20. Gene phrLS20 (brown arrow, phr) encodes a pre-pro-protein of 44 residues. This protein is subject to an export-maturation-import route. The mature pentapeptide inhibits activity of the RapLS20 anti-repressor protein. For simplicity, import of the mature peptide has been shown into the cell producing the peptide. Grey cylinders labeled sec and imp, respectively, indicate the secretion and import routes. Extracellular processing of the secreted peptide is symbolized by the brown interrupted rectangle. QS, quorum sensing.
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pgen-1003892-g008: Model of regulatory circuitry of pLS20 conjugation genes.A. Repressed state due to RcoLS20. Gene rcoLS20 (red arrow, rco) encoding the master repressor of conjugation genes RcoLS20 is divergently transcribed from the putative conjugation operon encompassing genes 28 to 74 (light blue arrows). RcoLS20 inhibits expression of the conjugation genes by repressing a promoter, Pc, located upstream of gene 28, the first gene of the putative conjugation operon (our unpublished results). B. Activation of conjugation by RapLS20 anti-repressor. Gene rapLS20 (green arrow, rap) encodes the anti-repressor of RcoLS20 leading to de-repression of the conjugation genes. C. Repressed state due to inactivation of RapLS20 by signaling peptide Phr*LS20. Gene phrLS20 (brown arrow, phr) encodes a pre-pro-protein of 44 residues. This protein is subject to an export-maturation-import route. The mature pentapeptide inhibits activity of the RapLS20 anti-repressor protein. For simplicity, import of the mature peptide has been shown into the cell producing the peptide. Grey cylinders labeled sec and imp, respectively, indicate the secretion and import routes. Extracellular processing of the secreted peptide is symbolized by the brown interrupted rectangle. QS, quorum sensing.

Mentions: A summary of the regulatory circuitry of the pLS20 conjugation genes is schematically presented in Figure 8. RcoLS20 is responsible for maintaining conjugation in the default “OFF” state by repressing the conjugation genes. RapLS20 can activate conjugation by relieving RcoLS20-mediated repression, but is only able to do so when its activity is not inhibited by the Phr*LS20 signaling peptide. Therefore, conjugation of the pLS20cat plasmid is strictly regulated by the Phr*LS20 peptide-mediated quorum sensing (QS) mechanism. QS is a common way by which bacteria communicate with one another using small and diffusible chemical signaling molecules. When the concentration of a signaling molecule reaches a certain “quorum”, bacteria respond by altering its gene expression profile at a (sub)population-wide scale (for review see, [49], [50]). Several cellular processes in both Gram+ positive and Gram- bacteria have been shown to be regulated by QS, among them the development of natural competence in B. subtilis and Streptococcus pneumonia, [30], [43], [50]. Here, we show that QS plays a role in HGT at another level by regulating expression of conjugation genes of plasmid pLS20. So far, QS has been reported to regulate conjugation genes of only a few other conjugative elements. These include the transfer of the tumor-inducing pTI plasmid of the Gram- Agrobacterium tumefaciens into plant cells. In this case, activation of conjugation requires two signaling peptides, one produced by the plant and the other by the donor cell [51]. Regulation of conjugation of the enterococcal plasmid pCF10, -and probably in a similar way pAD1-, also involves two signaling peptides, one produced by donor and the other by recipient cells. The two peptides compete for binding to a single transcriptional regulator, PrgX, and act antagonistically on conjugation. However, instead of being an activator, PrgX is a repressor. When PrgX is bound to the donor-produced signaling peptide the complex binds DNA and represses the conjugation genes. Conjugation genes become activated when recipient-produced signaling peptide replaces the donor-produced signaling peptide in the PrgX/peptide complex thereby inactivating the repressor activity of PrgX. Consequently, conjugation genes are activated by recipient produced signaling peptides [17]. Our results show that the QS mechanism to regulate conjugation genes of pLS20 differs in various aspects from those regulating conjugation of the pTi and pCF10/pAD1 plasmids. First, regulation of pLS20 conjugation genes involves not two but only one signaling peptide, Phr*LS20. Second, the signaling peptide does not act directly on the transcriptional regulator but instead regulates activity of another protein, RapLS20, which functions as an anti-repressor. And third, the signaling peptide does not function to activate conjugation genes but to return the conjugation system to the default “OFF” state by inhibiting the activity of the anti-repressor.


Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

Singh PK, Ramachandran G, Ramos-Ruiz R, Peiró-Pastor R, Abia D, Wu LJ, Meijer WJ - PLoS Genet. (2013)

Model of regulatory circuitry of pLS20 conjugation genes.A. Repressed state due to RcoLS20. Gene rcoLS20 (red arrow, rco) encoding the master repressor of conjugation genes RcoLS20 is divergently transcribed from the putative conjugation operon encompassing genes 28 to 74 (light blue arrows). RcoLS20 inhibits expression of the conjugation genes by repressing a promoter, Pc, located upstream of gene 28, the first gene of the putative conjugation operon (our unpublished results). B. Activation of conjugation by RapLS20 anti-repressor. Gene rapLS20 (green arrow, rap) encodes the anti-repressor of RcoLS20 leading to de-repression of the conjugation genes. C. Repressed state due to inactivation of RapLS20 by signaling peptide Phr*LS20. Gene phrLS20 (brown arrow, phr) encodes a pre-pro-protein of 44 residues. This protein is subject to an export-maturation-import route. The mature pentapeptide inhibits activity of the RapLS20 anti-repressor protein. For simplicity, import of the mature peptide has been shown into the cell producing the peptide. Grey cylinders labeled sec and imp, respectively, indicate the secretion and import routes. Extracellular processing of the secreted peptide is symbolized by the brown interrupted rectangle. QS, quorum sensing.
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pgen-1003892-g008: Model of regulatory circuitry of pLS20 conjugation genes.A. Repressed state due to RcoLS20. Gene rcoLS20 (red arrow, rco) encoding the master repressor of conjugation genes RcoLS20 is divergently transcribed from the putative conjugation operon encompassing genes 28 to 74 (light blue arrows). RcoLS20 inhibits expression of the conjugation genes by repressing a promoter, Pc, located upstream of gene 28, the first gene of the putative conjugation operon (our unpublished results). B. Activation of conjugation by RapLS20 anti-repressor. Gene rapLS20 (green arrow, rap) encodes the anti-repressor of RcoLS20 leading to de-repression of the conjugation genes. C. Repressed state due to inactivation of RapLS20 by signaling peptide Phr*LS20. Gene phrLS20 (brown arrow, phr) encodes a pre-pro-protein of 44 residues. This protein is subject to an export-maturation-import route. The mature pentapeptide inhibits activity of the RapLS20 anti-repressor protein. For simplicity, import of the mature peptide has been shown into the cell producing the peptide. Grey cylinders labeled sec and imp, respectively, indicate the secretion and import routes. Extracellular processing of the secreted peptide is symbolized by the brown interrupted rectangle. QS, quorum sensing.
Mentions: A summary of the regulatory circuitry of the pLS20 conjugation genes is schematically presented in Figure 8. RcoLS20 is responsible for maintaining conjugation in the default “OFF” state by repressing the conjugation genes. RapLS20 can activate conjugation by relieving RcoLS20-mediated repression, but is only able to do so when its activity is not inhibited by the Phr*LS20 signaling peptide. Therefore, conjugation of the pLS20cat plasmid is strictly regulated by the Phr*LS20 peptide-mediated quorum sensing (QS) mechanism. QS is a common way by which bacteria communicate with one another using small and diffusible chemical signaling molecules. When the concentration of a signaling molecule reaches a certain “quorum”, bacteria respond by altering its gene expression profile at a (sub)population-wide scale (for review see, [49], [50]). Several cellular processes in both Gram+ positive and Gram- bacteria have been shown to be regulated by QS, among them the development of natural competence in B. subtilis and Streptococcus pneumonia, [30], [43], [50]. Here, we show that QS plays a role in HGT at another level by regulating expression of conjugation genes of plasmid pLS20. So far, QS has been reported to regulate conjugation genes of only a few other conjugative elements. These include the transfer of the tumor-inducing pTI plasmid of the Gram- Agrobacterium tumefaciens into plant cells. In this case, activation of conjugation requires two signaling peptides, one produced by the plant and the other by the donor cell [51]. Regulation of conjugation of the enterococcal plasmid pCF10, -and probably in a similar way pAD1-, also involves two signaling peptides, one produced by donor and the other by recipient cells. The two peptides compete for binding to a single transcriptional regulator, PrgX, and act antagonistically on conjugation. However, instead of being an activator, PrgX is a repressor. When PrgX is bound to the donor-produced signaling peptide the complex binds DNA and represses the conjugation genes. Conjugation genes become activated when recipient-produced signaling peptide replaces the donor-produced signaling peptide in the PrgX/peptide complex thereby inactivating the repressor activity of PrgX. Consequently, conjugation genes are activated by recipient produced signaling peptides [17]. Our results show that the QS mechanism to regulate conjugation genes of pLS20 differs in various aspects from those regulating conjugation of the pTi and pCF10/pAD1 plasmids. First, regulation of pLS20 conjugation genes involves not two but only one signaling peptide, Phr*LS20. Second, the signaling peptide does not act directly on the transcriptional regulator but instead regulates activity of another protein, RapLS20, which functions as an anti-repressor. And third, the signaling peptide does not function to activate conjugation genes but to return the conjugation system to the default “OFF” state by inhibiting the activity of the anti-repressor.

Bottom Line: Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes.Ultimately, this peptide dictates the timing of conjugation.The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.

ABSTRACT
Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Show MeSH