Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for ...genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.
Integrative and conjugative elements (ICEs) are major contributors to genome plasticity in bacteria. ICEs reside integrated in the chromosome of a host bacterium and are passively propagated during ...chromosome replication and cell division. When activated, ICEs excise from the chromosome and may be transferred through the ICE-encoded conjugation machinery into a recipient cell. Integration into the chromosome of the new host generates a stable transconjugant. Although integration into the chromosome of a new host is critical for the stable acquisition of ICEs, few studies have directly investigated the molecular events that occur in recipient cells during generation of a stable transconjugant. We found that integration of ICEBs1, an ICE of Bacillus subtilis, occurred several generations after initial transfer to a new host. Premature integration in new hosts led to cell death and hence decreased fitness of the ICE and transconjugants. Host lethality due to premature integration was caused by rolling circle replication that initiated in the integrated ICEBs1 and extended into the host chromosome, resulting in catastrophic genome instability. Our results demonstrate that the timing of integration of an ICE is linked to cessation of autonomous replication of the ICE, and that perturbing this linkage leads to a decrease in ICE and host fitness due to a loss of viability of transconjugants. Linking integration to cessation of autonomous replication appears to be a conserved regulatory scheme for mobile genetic elements that both replicate and integrate into the chromosome of their host.
Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how ...these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SPß and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SPß, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SPß killing) was both necessary and sufficient for this protection. spbK inhibited production of SPß, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SPß gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.
Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) ...are modular mobile genetic elements integrated into a host genome and are passively propagated during chromosomal replication and cell division. Induction of ICE gene expression leads to excision, production of the conserved conjugation machinery (a type IV secretion system), and the potential to transfer DNA to appropriate recipients. ICEs typically contain cargo genes that are not usually related to the ICE life cycle and that confer phenotypes to host cells. We summarize the life cycle and discovery of ICEs, some of the regulatory mechanisms, and how the types of cargo have influenced our view of ICEs. We discuss how ICEs can acquire new cargo genes and describe challenges to the field and various perspectives on ICE biology.
DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., ...DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.
DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separation of these two processes leads to their conflict, and failure to ...deal with this conflict can result in genome alterations and reduced fitness. In recent years major advances have been made in understanding how cells avoid conflicts between replication and transcription and how such conflicts are resolved when they do occur. In this Review, we summarize these findings, which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.
Abstract
Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer by conjugation to recipient cells. Some ICEs integrate into a unique site in the genome of their ...hosts. We studied quantitatively the process by which an ICE searches for its unique integration site in the Bacillus subtilis chromosome. We followed the motion of both ICEBs1 and the chromosomal integration site in real time within individual cells. ICEBs1 exhibited a wide spectrum of dynamical behaviors, ranging from rapid sub-diffusive displacements crisscrossing the cell, to kinetically trapped states. The chromosomal integration site moved sub-diffusively and exhibited pronounced dynamical asymmetry between longitudinal and transversal motions, highlighting the role of chromosomal structure and the heterogeneity of the bacterial interior in the search. The successful search for and subsequent recombination into the integration site is a key step in the acquisition of integrating mobile genetic elements. Our findings provide new insights into intracellular transport processes involving large DNA molecules.
Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to host cells, including antibiotic resistance, metabolic ...capabilities, or pathogenic determinants. ICEs can also have negative effects on host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. Using this reporter, we found that cell division was arrested in cells of Bacillus subtilis and Enterococcus faecalis (a natural host for Tn916) that contained an activated (excised) Tn916. Furthermore, most of the cells with the activated Tn916 subsequently died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause growth defects in B. subtilis and identified a single gene, yqaR, that is in a defective phage (skin) in the B. subtilis chromosome that was required for this phenotype. These three genes were only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.
DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to ...the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.
The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to ...extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.
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