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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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Genetic map of pLS20cat.(Putative) genes are numbered. Gene 1 corresponds to the homologue of gene 1 of the related Bacillus pumilus NRS576 plasmid p576 [37]. The positions and the lengths of the (putative) genes are indicated by arrows. Rightward and leftward oriented genes are indicated in purple and orange, respectively. Putative Rho-independent transcriptional terminators are indicated with green hairpin structures. The origin of replication region and the gene conferring resistance to chloramphenicol are labeled with green rectangles. The DNA region containing the chloramphenicol gene was cloned into the unique SalI site located in pLS20 gene 13 [24]. The sequences flanking the Cm resistance cassette coding for the N- and C-terminal regions of gene 13 are labeled 13-N and 13-C, respectively. The putative conjugation operon encompassing genes 28 to 74, is highlighted by a blue background. Genes showing significant homology with genes reported to be involved in conjugation in other systems are shown in black. Recently, the complete pLS20cat sequence has been deposited by Itaya,M., et al. (Mitsuhiro Itaya Keio University, Japan) in public database under accession numbers NC_015148.1 and AB615352.1. pLS20cat gene 25, according to our nomenclature, corresponds to gene 001 of the deposited sequence. Due to differences in annotation we prefer to maintain our nomenclature.
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pgen-1003892-g002: Genetic map of pLS20cat.(Putative) genes are numbered. Gene 1 corresponds to the homologue of gene 1 of the related Bacillus pumilus NRS576 plasmid p576 [37]. The positions and the lengths of the (putative) genes are indicated by arrows. Rightward and leftward oriented genes are indicated in purple and orange, respectively. Putative Rho-independent transcriptional terminators are indicated with green hairpin structures. The origin of replication region and the gene conferring resistance to chloramphenicol are labeled with green rectangles. The DNA region containing the chloramphenicol gene was cloned into the unique SalI site located in pLS20 gene 13 [24]. The sequences flanking the Cm resistance cassette coding for the N- and C-terminal regions of gene 13 are labeled 13-N and 13-C, respectively. The putative conjugation operon encompassing genes 28 to 74, is highlighted by a blue background. Genes showing significant homology with genes reported to be involved in conjugation in other systems are shown in black. Recently, the complete pLS20cat sequence has been deposited by Itaya,M., et al. (Mitsuhiro Itaya Keio University, Japan) in public database under accession numbers NC_015148.1 and AB615352.1. pLS20cat gene 25, according to our nomenclature, corresponds to gene 001 of the deposited sequence. Due to differences in annotation we prefer to maintain our nomenclature.

Mentions: The observation that efficient conjugation occurred only during a short time window raised the possibility that conjugation is kept in the default “OFF” state by a transcriptional repressor protein, and is switched on only in a certain period during growth when the repressor is inactivated. To identify a possible conjugation repressor gene we sequenced and annotated pLS20cat, and used this information to construct a genetic map of pLS20cat (Figure 2). The following features identified gene 27c as a possible candidate encoding a conjugation repressor. First, in silico analysis indicated that it encodes an Xre-type, transcriptional regulator with a Helix-Turn-Helix (HTH) domain in its N-terminal region (see Figure S1). Second, gene 27c is located immediately upstream of a divergently oriented putative conjugation operon spanning genes 28 to 74. Several of the genes in the 28 to 74 region are predicted to be homologues of essential conjugation genes present on other conjugative plasmids, and homologues of essential conjugation genes are not found outside this region of pLS20cat (see Figure 2). Table S1 gives an overview of the comparative analysis of genes in this region that includes details on the putative translation start sites.


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)

Genetic map of pLS20cat.(Putative) genes are numbered. Gene 1 corresponds to the homologue of gene 1 of the related Bacillus pumilus NRS576 plasmid p576 [37]. The positions and the lengths of the (putative) genes are indicated by arrows. Rightward and leftward oriented genes are indicated in purple and orange, respectively. Putative Rho-independent transcriptional terminators are indicated with green hairpin structures. The origin of replication region and the gene conferring resistance to chloramphenicol are labeled with green rectangles. The DNA region containing the chloramphenicol gene was cloned into the unique SalI site located in pLS20 gene 13 [24]. The sequences flanking the Cm resistance cassette coding for the N- and C-terminal regions of gene 13 are labeled 13-N and 13-C, respectively. The putative conjugation operon encompassing genes 28 to 74, is highlighted by a blue background. Genes showing significant homology with genes reported to be involved in conjugation in other systems are shown in black. Recently, the complete pLS20cat sequence has been deposited by Itaya,M., et al. (Mitsuhiro Itaya Keio University, Japan) in public database under accession numbers NC_015148.1 and AB615352.1. pLS20cat gene 25, according to our nomenclature, corresponds to gene 001 of the deposited sequence. Due to differences in annotation we prefer to maintain our nomenclature.
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Related In: Results  -  Collection

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pgen-1003892-g002: Genetic map of pLS20cat.(Putative) genes are numbered. Gene 1 corresponds to the homologue of gene 1 of the related Bacillus pumilus NRS576 plasmid p576 [37]. The positions and the lengths of the (putative) genes are indicated by arrows. Rightward and leftward oriented genes are indicated in purple and orange, respectively. Putative Rho-independent transcriptional terminators are indicated with green hairpin structures. The origin of replication region and the gene conferring resistance to chloramphenicol are labeled with green rectangles. The DNA region containing the chloramphenicol gene was cloned into the unique SalI site located in pLS20 gene 13 [24]. The sequences flanking the Cm resistance cassette coding for the N- and C-terminal regions of gene 13 are labeled 13-N and 13-C, respectively. The putative conjugation operon encompassing genes 28 to 74, is highlighted by a blue background. Genes showing significant homology with genes reported to be involved in conjugation in other systems are shown in black. Recently, the complete pLS20cat sequence has been deposited by Itaya,M., et al. (Mitsuhiro Itaya Keio University, Japan) in public database under accession numbers NC_015148.1 and AB615352.1. pLS20cat gene 25, according to our nomenclature, corresponds to gene 001 of the deposited sequence. Due to differences in annotation we prefer to maintain our nomenclature.
Mentions: The observation that efficient conjugation occurred only during a short time window raised the possibility that conjugation is kept in the default “OFF” state by a transcriptional repressor protein, and is switched on only in a certain period during growth when the repressor is inactivated. To identify a possible conjugation repressor gene we sequenced and annotated pLS20cat, and used this information to construct a genetic map of pLS20cat (Figure 2). The following features identified gene 27c as a possible candidate encoding a conjugation repressor. First, in silico analysis indicated that it encodes an Xre-type, transcriptional regulator with a Helix-Turn-Helix (HTH) domain in its N-terminal region (see Figure S1). Second, gene 27c is located immediately upstream of a divergently oriented putative conjugation operon spanning genes 28 to 74. Several of the genes in the 28 to 74 region are predicted to be homologues of essential conjugation genes present on other conjugative plasmids, and homologues of essential conjugation genes are not found outside this region of pLS20cat (see Figure 2). Table S1 gives an overview of the comparative analysis of genes in this region that includes details on the putative translation start sites.

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