Limits...
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.

Show MeSH

Related in: MedlinePlus

Phr*LS20 pentapeptide inhibits conjugation in an opp dependent manner.A. Effects of synthetic Phr* peptide on conjugation in the wild type and an opp deficient background. Conjugation efficiencies of pLS20cat were determined at late exponential growth phase using as recipient strain PKS7, and as donor either strain PKS11 (wild type, black bars) or PKS98 (oppA, grey bars). Diluted overnight grown cultures of donor cells were split in two, and Phr*LS20 pentapeptide was added to a final concentration of 6 µM to one of the cultures and equal volume of the peptide buffer to the other. B. Conjugation kinetics of pLS20cat and pLS20phr. Conjugation kinetics was determined as described in Materials and Methods using PKS7 as recipient strain and PKS14 (pLS20cat) or PKS117 (pLS20phr) as donor strains. t = 0 corresponds to the end of the exponential growth phase. Both donor strains contain an ectopic copy of rcoLS20 under the IPTG inducible Pspank promoter at the chromosomal amyE locus. Overnight cultures of donor cells were grown in the presence of 1 mM IPTG and diluted in fresh pre-warmed LB medium without IPTG. C. Conjugation kinetics of pLS20cat after re-dilution of the donor cell culture. Conjugation kinetics using PKS7 and PKS11 as recipient and donor strains, respectively, was determined as described in Materials and Methods with the following modification. Overnight cultures were diluted, grown until late exponential growth phase (OD600 = 0,8), and diluted again (to OD600 = 0.05) before starting the experiment. B and C. t = 0 corresponds to the end of the exponential growth phase.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814332&req=5

pgen-1003892-g006: Phr*LS20 pentapeptide inhibits conjugation in an opp dependent manner.A. Effects of synthetic Phr* peptide on conjugation in the wild type and an opp deficient background. Conjugation efficiencies of pLS20cat were determined at late exponential growth phase using as recipient strain PKS7, and as donor either strain PKS11 (wild type, black bars) or PKS98 (oppA, grey bars). Diluted overnight grown cultures of donor cells were split in two, and Phr*LS20 pentapeptide was added to a final concentration of 6 µM to one of the cultures and equal volume of the peptide buffer to the other. B. Conjugation kinetics of pLS20cat and pLS20phr. Conjugation kinetics was determined as described in Materials and Methods using PKS7 as recipient strain and PKS14 (pLS20cat) or PKS117 (pLS20phr) as donor strains. t = 0 corresponds to the end of the exponential growth phase. Both donor strains contain an ectopic copy of rcoLS20 under the IPTG inducible Pspank promoter at the chromosomal amyE locus. Overnight cultures of donor cells were grown in the presence of 1 mM IPTG and diluted in fresh pre-warmed LB medium without IPTG. C. Conjugation kinetics of pLS20cat after re-dilution of the donor cell culture. Conjugation kinetics using PKS7 and PKS11 as recipient and donor strains, respectively, was determined as described in Materials and Methods with the following modification. Overnight cultures were diluted, grown until late exponential growth phase (OD600 = 0,8), and diluted again (to OD600 = 0.05) before starting the experiment. B and C. t = 0 corresponds to the end of the exponential growth phase.

Mentions: Many rap genes are transcriptionally coupled to a downstream-located phr gene. The small phr genes encode a product that, after being subjected to an export-import-maturation process, produces a mature penta- or hexapeptide that inhibits the activity of its cognate Rap protein. A putative phr gene, phrLS20, is located immediately downstream of rapLS20. The stop/start codons of these genes overlap and hence phrLS20 is translationally coupled to rapLS20, a situation that is similar to those observed for some other rap-phr cassettes. Inspection of the deduced protein sequence suggests that phrLS20 indeed encodes a typical pre-pro-peptide. The 44 residue gene product is predicted to contain an N-terminal signal peptide, a conserved motif upstream of its predicted maturation cleavage site, as well as conserved residues within the putative mature peptide [25], [43]. Based on this, the mature phrLS20–derived peptide is predicted to correspond to the five C-terminal residues of Phr*LS20, “QKGMY”, which we will refer to as Phr*LS20. To test a possible effect we determined conjugation efficiencies at the end of the exponential growth phase in the absence or presence of synthetic “QKGMY” peptide. The results presented in Figure 6A show that the presence of synthetic Phr*LS20 in the medium greatly reduced the maximum level of conjugation. These results support the view that Phr*LS20 inhibits RapLS20–mediated de-repression of the conjugation genes. Conjugation efficiency did not alter significantly in the presence of another pentapeptide “EKAII”, demonstrating the specificity of the Phr*LS20 (not shown). The “EKAII” peptide is the predicted mature Phr*576 peptide encoded by a rap-phr cassette located on the related p576 plasmid [37].


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)

Phr*LS20 pentapeptide inhibits conjugation in an opp dependent manner.A. Effects of synthetic Phr* peptide on conjugation in the wild type and an opp deficient background. Conjugation efficiencies of pLS20cat were determined at late exponential growth phase using as recipient strain PKS7, and as donor either strain PKS11 (wild type, black bars) or PKS98 (oppA, grey bars). Diluted overnight grown cultures of donor cells were split in two, and Phr*LS20 pentapeptide was added to a final concentration of 6 µM to one of the cultures and equal volume of the peptide buffer to the other. B. Conjugation kinetics of pLS20cat and pLS20phr. Conjugation kinetics was determined as described in Materials and Methods using PKS7 as recipient strain and PKS14 (pLS20cat) or PKS117 (pLS20phr) as donor strains. t = 0 corresponds to the end of the exponential growth phase. Both donor strains contain an ectopic copy of rcoLS20 under the IPTG inducible Pspank promoter at the chromosomal amyE locus. Overnight cultures of donor cells were grown in the presence of 1 mM IPTG and diluted in fresh pre-warmed LB medium without IPTG. C. Conjugation kinetics of pLS20cat after re-dilution of the donor cell culture. Conjugation kinetics using PKS7 and PKS11 as recipient and donor strains, respectively, was determined as described in Materials and Methods with the following modification. Overnight cultures were diluted, grown until late exponential growth phase (OD600 = 0,8), and diluted again (to OD600 = 0.05) before starting the experiment. B and C. t = 0 corresponds to the end of the exponential growth phase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814332&req=5

pgen-1003892-g006: Phr*LS20 pentapeptide inhibits conjugation in an opp dependent manner.A. Effects of synthetic Phr* peptide on conjugation in the wild type and an opp deficient background. Conjugation efficiencies of pLS20cat were determined at late exponential growth phase using as recipient strain PKS7, and as donor either strain PKS11 (wild type, black bars) or PKS98 (oppA, grey bars). Diluted overnight grown cultures of donor cells were split in two, and Phr*LS20 pentapeptide was added to a final concentration of 6 µM to one of the cultures and equal volume of the peptide buffer to the other. B. Conjugation kinetics of pLS20cat and pLS20phr. Conjugation kinetics was determined as described in Materials and Methods using PKS7 as recipient strain and PKS14 (pLS20cat) or PKS117 (pLS20phr) as donor strains. t = 0 corresponds to the end of the exponential growth phase. Both donor strains contain an ectopic copy of rcoLS20 under the IPTG inducible Pspank promoter at the chromosomal amyE locus. Overnight cultures of donor cells were grown in the presence of 1 mM IPTG and diluted in fresh pre-warmed LB medium without IPTG. C. Conjugation kinetics of pLS20cat after re-dilution of the donor cell culture. Conjugation kinetics using PKS7 and PKS11 as recipient and donor strains, respectively, was determined as described in Materials and Methods with the following modification. Overnight cultures were diluted, grown until late exponential growth phase (OD600 = 0,8), and diluted again (to OD600 = 0.05) before starting the experiment. B and C. t = 0 corresponds to the end of the exponential growth phase.
Mentions: Many rap genes are transcriptionally coupled to a downstream-located phr gene. The small phr genes encode a product that, after being subjected to an export-import-maturation process, produces a mature penta- or hexapeptide that inhibits the activity of its cognate Rap protein. A putative phr gene, phrLS20, is located immediately downstream of rapLS20. The stop/start codons of these genes overlap and hence phrLS20 is translationally coupled to rapLS20, a situation that is similar to those observed for some other rap-phr cassettes. Inspection of the deduced protein sequence suggests that phrLS20 indeed encodes a typical pre-pro-peptide. The 44 residue gene product is predicted to contain an N-terminal signal peptide, a conserved motif upstream of its predicted maturation cleavage site, as well as conserved residues within the putative mature peptide [25], [43]. Based on this, the mature phrLS20–derived peptide is predicted to correspond to the five C-terminal residues of Phr*LS20, “QKGMY”, which we will refer to as Phr*LS20. To test a possible effect we determined conjugation efficiencies at the end of the exponential growth phase in the absence or presence of synthetic “QKGMY” peptide. The results presented in Figure 6A show that the presence of synthetic Phr*LS20 in the medium greatly reduced the maximum level of conjugation. These results support the view that Phr*LS20 inhibits RapLS20–mediated de-repression of the conjugation genes. Conjugation efficiency did not alter significantly in the presence of another pentapeptide “EKAII”, demonstrating the specificity of the Phr*LS20 (not shown). The “EKAII” peptide is the predicted mature Phr*576 peptide encoded by a rap-phr cassette located on the related p576 plasmid [37].

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
Related in: MedlinePlus