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Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Wright LD, Johnson CM, Grossman AD - PLoS Genet. (2015)

Bottom Line: Several functional assays confirmed Sso activity.We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host.Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

No MeSH data available.


Related in: MedlinePlus

Model of ICE integration with an sso downstream (A) and upstream (B) of oriT.For all ICEs, excision from the chromosome yields a dsDNA circle (concentric black circles) with an origin of transfer (oriT, black slash mark) and the attachment site, att (filled diamond). Prior to conjugation, relaxase (black oval) nicks at oriT and covalently attaches to the 5’ end. The relaxase, attached to ssDNA (gray solid line) containing the sso (gray rectangle), is transferred to recipients. In the transconjugant, the relaxase catalyzes strand ligation and formation of a ssDNA circle [reviewed in 21]. Based on known mechanisms of site-specific recombination, the att site must be double-stranded in order for the ICE to integrate into the recipient chromosome (parallel black lines). If the att site becomes dsDNA before the nicked DNA is recircularized, and if this incomplete ICE were to integrate into the chromosome, then a double strand break in the chromosome would be created. (A) The sso in an ICE is downstream from oriT. Second strand synthesis (dotted gray line) cannot proceed through oriT until the linear DNA becomes circularized. Once the att site in ICE becomes double-stranded, site-specific recombination into the chromosome can occur, and the product will be a fully integrated element with an intact genome. B) The sso in an ICE is upstream from oriT. Second strand synthesis (gray dotted line) can occur on the linear DNA, and it is possible that the att site becomes double-stranded before the ICE circularizes. If this form of ICE is capable of undergoing site-specific recombination, then integration of this linear dsDNA into the chromosome will yield a double-stranded break (DSB).
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pgen.1005556.g008: Model of ICE integration with an sso downstream (A) and upstream (B) of oriT.For all ICEs, excision from the chromosome yields a dsDNA circle (concentric black circles) with an origin of transfer (oriT, black slash mark) and the attachment site, att (filled diamond). Prior to conjugation, relaxase (black oval) nicks at oriT and covalently attaches to the 5’ end. The relaxase, attached to ssDNA (gray solid line) containing the sso (gray rectangle), is transferred to recipients. In the transconjugant, the relaxase catalyzes strand ligation and formation of a ssDNA circle [reviewed in 21]. Based on known mechanisms of site-specific recombination, the att site must be double-stranded in order for the ICE to integrate into the recipient chromosome (parallel black lines). If the att site becomes dsDNA before the nicked DNA is recircularized, and if this incomplete ICE were to integrate into the chromosome, then a double strand break in the chromosome would be created. (A) The sso in an ICE is downstream from oriT. Second strand synthesis (dotted gray line) cannot proceed through oriT until the linear DNA becomes circularized. Once the att site in ICE becomes double-stranded, site-specific recombination into the chromosome can occur, and the product will be a fully integrated element with an intact genome. B) The sso in an ICE is upstream from oriT. Second strand synthesis (gray dotted line) can occur on the linear DNA, and it is possible that the att site becomes double-stranded before the ICE circularizes. If this form of ICE is capable of undergoing site-specific recombination, then integration of this linear dsDNA into the chromosome will yield a double-stranded break (DSB).

Mentions: The location of sso1 relative to oriT in ICEBs1 could increase the probability of successful chromosome re-integration. sso1 in ICEBs1 is downstream of oriT, the double-stranded origin of replication (dso). In contrast, the sso in most RCR plasmids is upstream of the dso [23], although there are some exceptions [e.g., 28]. The positioning of an sso upstream of the dso in plasmids ensures that the sso is not single-stranded (and thus active) until leading strand synthesis from the dso is almost complete. However, the location of ICEBs1 sso1 relative to oriT ensures that the attachment site in the circular ICEBs1 (attICE, previously referred to as attP) is not double-stranded (and thus a substrate for site-specific recombination into the chromosome) until ligation and recircularization at the nic site (in oriT) occurs. Initiation of second strand synthesis from an sso upstream of oriT could result in replication of attICE before recircularization, and integration could result in a double-strand break in the chromosome (Fig 8). Thus, the location of sso's in ICEs may be an adaptation to the ICE lifecycle to prevent premature integration and possible damage to the host.


Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Wright LD, Johnson CM, Grossman AD - PLoS Genet. (2015)

Model of ICE integration with an sso downstream (A) and upstream (B) of oriT.For all ICEs, excision from the chromosome yields a dsDNA circle (concentric black circles) with an origin of transfer (oriT, black slash mark) and the attachment site, att (filled diamond). Prior to conjugation, relaxase (black oval) nicks at oriT and covalently attaches to the 5’ end. The relaxase, attached to ssDNA (gray solid line) containing the sso (gray rectangle), is transferred to recipients. In the transconjugant, the relaxase catalyzes strand ligation and formation of a ssDNA circle [reviewed in 21]. Based on known mechanisms of site-specific recombination, the att site must be double-stranded in order for the ICE to integrate into the recipient chromosome (parallel black lines). If the att site becomes dsDNA before the nicked DNA is recircularized, and if this incomplete ICE were to integrate into the chromosome, then a double strand break in the chromosome would be created. (A) The sso in an ICE is downstream from oriT. Second strand synthesis (dotted gray line) cannot proceed through oriT until the linear DNA becomes circularized. Once the att site in ICE becomes double-stranded, site-specific recombination into the chromosome can occur, and the product will be a fully integrated element with an intact genome. B) The sso in an ICE is upstream from oriT. Second strand synthesis (gray dotted line) can occur on the linear DNA, and it is possible that the att site becomes double-stranded before the ICE circularizes. If this form of ICE is capable of undergoing site-specific recombination, then integration of this linear dsDNA into the chromosome will yield a double-stranded break (DSB).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595007&req=5

pgen.1005556.g008: Model of ICE integration with an sso downstream (A) and upstream (B) of oriT.For all ICEs, excision from the chromosome yields a dsDNA circle (concentric black circles) with an origin of transfer (oriT, black slash mark) and the attachment site, att (filled diamond). Prior to conjugation, relaxase (black oval) nicks at oriT and covalently attaches to the 5’ end. The relaxase, attached to ssDNA (gray solid line) containing the sso (gray rectangle), is transferred to recipients. In the transconjugant, the relaxase catalyzes strand ligation and formation of a ssDNA circle [reviewed in 21]. Based on known mechanisms of site-specific recombination, the att site must be double-stranded in order for the ICE to integrate into the recipient chromosome (parallel black lines). If the att site becomes dsDNA before the nicked DNA is recircularized, and if this incomplete ICE were to integrate into the chromosome, then a double strand break in the chromosome would be created. (A) The sso in an ICE is downstream from oriT. Second strand synthesis (dotted gray line) cannot proceed through oriT until the linear DNA becomes circularized. Once the att site in ICE becomes double-stranded, site-specific recombination into the chromosome can occur, and the product will be a fully integrated element with an intact genome. B) The sso in an ICE is upstream from oriT. Second strand synthesis (gray dotted line) can occur on the linear DNA, and it is possible that the att site becomes double-stranded before the ICE circularizes. If this form of ICE is capable of undergoing site-specific recombination, then integration of this linear dsDNA into the chromosome will yield a double-stranded break (DSB).
Mentions: The location of sso1 relative to oriT in ICEBs1 could increase the probability of successful chromosome re-integration. sso1 in ICEBs1 is downstream of oriT, the double-stranded origin of replication (dso). In contrast, the sso in most RCR plasmids is upstream of the dso [23], although there are some exceptions [e.g., 28]. The positioning of an sso upstream of the dso in plasmids ensures that the sso is not single-stranded (and thus active) until leading strand synthesis from the dso is almost complete. However, the location of ICEBs1 sso1 relative to oriT ensures that the attachment site in the circular ICEBs1 (attICE, previously referred to as attP) is not double-stranded (and thus a substrate for site-specific recombination into the chromosome) until ligation and recircularization at the nic site (in oriT) occurs. Initiation of second strand synthesis from an sso upstream of oriT could result in replication of attICE before recircularization, and integration could result in a double-strand break in the chromosome (Fig 8). Thus, the location of sso's in ICEs may be an adaptation to the ICE lifecycle to prevent premature integration and possible damage to the host.

Bottom Line: Several functional assays confirmed Sso activity.We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host.Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

No MeSH data available.


Related in: MedlinePlus