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Tn917 targets the region where DNA replication terminates in Bacillus subtilis, highlighting a difference in chromosome processing in the firmicutes.

ABSTRACT: The bacterial transposon Tn917 inserts preferentially in the terminus region of some members of the Firmicutes. To determine what molecular process was being targeted by the element, we analyzed Tn917 target site selection in Bacillus subtilis. We find that Tn917 insertions accumulate around the central terminators, terI and terII, in wild-type cells with or without the SPbeta lysogen. Highly focused targeting around terI and terII requires the trans-acting termination protein RTP, but it is unaffected in strains compromised in dimer resolution or chromosome translocation. This work indicates that Tn917 is sensitive to differences in DNA replication termination between the Firmicutes.

SUBMITTER: Shi Q

PROVIDER: S-EPMC2786600 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Tn917 targets the region where DNA replication terminates in Bacillus subtilis, highlighting a difference in chromosome processing in the firmicutes.

Shi Qiaojuan Q Huguet-Tapia Jose C JC Peters Joseph E JE

Journal of bacteriology 20091009 24

The bacterial transposon Tn917 inserts preferentially in the terminus region of some members of the Firmicutes. To determine what molecular process was being targeted by the element, we analyzed Tn917 target site selection in Bacillus subtilis. We find that Tn917 insertions accumulate around the central terminators, terI and terII, in wild-type cells with or without the SPbeta lysogen. Highly focused targeting around terI and terII requires the trans-acting termination protein RTP, but it is una ...[more]

PMID: 19820088

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Project description:UnlabelledEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.ImportanceHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

Project description:BackgroundBacillus subtilis is one of the best-characterized organisms in Gram-positive bacteria. It represents a paradigm of gene regulation in bacteria due its complex life style (which could involve a transition between stages as diverse as vegetative cell and spore formation). In order to gain insight into the organization and evolution of the B. subtilis regulatory network and to provide an alternative framework for further studies in bacteria, we identified and analyzed its repertoire of DNA-binding transcription factors in terms of their abundance, family distribution and regulated genes.ResultsA collection of 237 DNA-binding Transcription Factors (TFs) was identified in B. subtilis, half of them with experimental evidence. 59% of them were predicted to be repressors, 17% activators, 17% were putatively identified as dual regulatory proteins and the remaining 6.3% could not be associated with a regulatory role. From this collection 56 TFs were found to be autoregulated, most of them negatively, though a significant proportion of positive feedback circuits were also identified. TFs were clustered into 51 regulatory protein families and then traced on 58 genomes from Firmicutes to detect their presence. From this analysis three families were found conserved in all the Firmicutes; fifteen families were distributed in all Firmicutes except in the phyla Mollicutes; two were constrained to Bacillales and finally two families were found to be specific to B. subtilis, due to their specie specific distribution. Repression seems to be the most common regulatory mechanism in Firmicutes due to the high proportion of repressors in the detected collection in these genomes. In addition, six global regulators were defined in B. subtilis based on the number and function of their regulated genes.ConclusionIn this work we identified and described the characteristics associated to the repertoire of DNA-binding TFs in B. subtilis. We also quantified their abundance, family distribution, and regulatory roles in the context of Firmicutes. This work should not only contribute to our understanding of the regulation of gene expression in bacteria from the perspective of B. subtilis but also provide us the basis for comprehensive modeling of transcriptional regulatory networks in Firmicutes.

Dataset Information

Tn917 targets the region where DNA replication terminates in Bacillus subtilis, highlighting a difference in chromosome processing in the firmicutes.

Publications

Tn917 targets the region where DNA replication terminates in Bacillus subtilis, highlighting a difference in chromosome processing in the firmicutes.

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