Timing of integration into the chromosome is critical for the fitness of an integrative and conjugative element and its bacterial host.

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.

Target search by an imported conjugative DNA element for a unique integration site along a bacterial chromosome during horizontal gene transfer.

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.

Beneficial and detrimental genes in the cellular response to replication arrest.

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.

TnSmu1 is a functional integrative and conjugative element in Streptococcus mutans that when expressed causes growth arrest of host bacteria.

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3' end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation of TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria.

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.

Multiple mechanisms for overcoming lethal over-initiation of DNA replication.

DNA replication is highly regulated and primarily controlled at the step of initiation. In bacteria, the replication initiator DnaA and the origin of replication oriC are the primary targets of regulation. Perturbations that increase or decrease replication initiation can cause a decrease in cell fitness. We found that multiple mechanisms, including an increase in replication elongation and a decrease in replication initiation, can compensate for lethal over-initiation. We found that in Bacillus subtilis, under conditions of rapid growth, loss of yabA, a negative regulator of replication initiation, caused a synthetic lethal phenotype when combined with the dnaA1 mutation that also causes replication over-initiation. We isolated several classes of suppressors that restored viability to dnaA1 ∆yabA double mutants. Some suppressors (relA, nrdR) stimulated replication elongation. Others (dnaC, cshA) caused a decrease in replication initiation. One class of suppressors decreased replication initiation in the dnaA1 ∆yabA mutant by causing a decrease in the amount of the replicative helicase, DnaC. We found that decreased levels of helicase in otherwise wild-type cells were sufficient to decrease replication initiation during rapid growth, indicating that the replicative helicase is limiting for replication initiation. Our results highlight the multiple mechanisms cells use to regulate DNA replication.

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies.

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.

A CRISPR interference screen reveals a role for cell wall teichoic acids in conjugation in Bacillus subtilis.

Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by active conjugation machines.

Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage.

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.

CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

A mobile genetic element increases bacterial host fitness by manipulating development.

Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes. Uncovering strategies used by mobile genetic elements to benefit host cells is crucial for understanding their stability and spread in populations. We describe a benefit that ICEBs1, an integrative and conjugative element of Bacillus subtilis, provides to its host cells. Activation of ICEBs1 conferred a frequency-dependent selective advantage to host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to inhibition of biofilm-associated gene expression and delayed sporulation by ICEBs1-containing cells, enabling them to exploit their neighbors and grow more prior to development. A single ICEBs1 gene, devI (formerly ydcO), was both necessary and sufficient for inhibition of development. Manipulation of host developmental programs allows ICEBs1 to increase host fitness, thereby increasing propagation of the element.

Many bacteria can 'have sex' ­ that is, they can share their genetic information and trade off segments of DNA. While these mobile genetic elements can be parasites that use the resources of their host to make more of themselves, some carry useful genes which, for example, help bacteria to fight off antibiotics. Integrative and conjugative elements (or ICEs) are a type of mobile segments that normally stay inside the genetic information of their bacterial host but can sometimes replicate and be pumped out to another cell. ICEBs1 for instance, is an element found in the common soil bacterium Bacillus subtilis. Scientists know that ICEBs1 can rapidly spread in biofilms ­ the slimly, crowded communities where bacteria live tightly connected ­ but it is still unclear whether it helps or hinders its hosts. Using genetic manipulations and tracking the survival of different groups of cells, Jones et al. show that carrying ICEBs1 confers an advantage under many conditions. When B. subtilis forms biofilms, the presence of the devI gene in ICEBs1 helps the cells to delay the production of the costly mucus that keeps bacteria together, allowing the organisms to 'cheat' for a little while and benefit from the tight-knit community without contributing to it. As nutrients become scarce in biofilms, the gene also allows the bacteria to grow for longer before they start to form spores ­ the dormant bacterial form that can weather difficult conditions. Mobile elements can carry genes that make bacteria resistant to antibiotics, harmful to humans, or able to use new food sources; they could even be used to artificially introduce genes of interest in these cells. The work by Jones et al. helps to understand the way these elements influence the fate of their host, providing insight into how they could be harnessed for the benefit of human health.

Specificity and Selective Advantage of an Exclusion System in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Integrative and conjugative elements (ICEs) are mobile genetic elements capable of transferring their own and other DNA. They contribute to the spread of antibiotic resistance and other important traits for bacterial evolution. Exclusion is a mechanism used by many conjugative plasmids and a few ICEs to prevent their host cell from acquiring a second copy of the cognate element. ICEBs1 of Bacillus subtilis has an exclusion mechanism whereby the exclusion protein YddJ in a potential recipient inhibits the activity of the ICEBs1-encoded conjugation machinery in a potential donor. The target of YddJ-mediated exclusion is the conjugation protein ConG (a VirB6 homolog). Here, we defined the regions of YddJ and ConG that confer exclusion specificity and determined the importance of exclusion to host cells. Using chimeras that had parts of ConG from ICEBs1 and the closely related ICEBat1, we identified a putative extracellular loop of ConG that conferred specificity for exclusion by the cognate YddJ. Using chimeras of YddJ from ICEBs1 and ICEBat1, we identified two regions in YddJ needed for exclusion specificity. We also found that YddJ-mediated exclusion reduced the death of donor cells following conjugation into recipients. Donor death was dependent on the ability of transconjugants to themselves become donors and was reduced under osmoprotective conditions, indicating that death was likely due to alterations in the donor cell envelope caused by excessive conjugation. We postulate that elements that can have high frequencies of transfer likely evolved exclusion mechanisms to protect the host cells from excessive death.IMPORTANCE Horizontal gene transfer is a driving force in bacterial evolution, responsible for the spread of many traits, including antibiotic and heavy metal resistance. Conjugation, one type of horizontal gene transfer, involves DNA transfer from donor to recipient cells through conjugation machinery and direct cell-cell contact. Exclusion mechanisms allow conjugative elements to prevent their host from acquiring additional copies of the element and are highly specific, enabling hosts to acquire heterologous elements. We defined regions of the exclusion protein and its target in the conjugation machinery that convey high specificity of exclusion. We found that exclusion protects donors from cell death during periods of high transfer. This is likely important for the element to enter new populations of cells.

Identification, characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis.

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram-negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram-positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1-encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion-resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria.

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-ß-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis.

DnaA is the widely conserved bacterial AAA+ ATPase that functions as both the replication initiator and a transcription factor. In many organisms, DnaA controls expression of its own gene and likely several others during growth and in response to replication stress. To evaluate the effects of DnaA on gene expression, separate from its role in replication initiation, we analyzed changes in mRNA levels in Bacillus subtilis cells with and without dnaA, using engineered strains in which dnaA is not essential. We found that dnaA was required for many of the changes in gene expression in response to replication stress. We also found that dnaA indirectly affected expression of several regulons during growth, including those controlled by the transcription factors Spo0A, AbrB, PhoP, SinR, RemA, Rok and YvrH. These effects were largely mediated by the effects of DnaA on expression of sda. DnaA activates transcription of sda, and Sda inhibits histidine protein kinases required for activation of the transcription factor Spo0A. We also found that loss of dnaA caused a decrease in the development of genetic competence. Together, our results indicate that DnaA plays an important role in modulating cell physiology, separate from its role in replication initiation.

A Mobile Element in mutS Drives Hypermutation in a Marine Vibrio.

Bacteria face a trade-off between genetic fidelity, which reduces deleterious mistakes in the genome, and genetic innovation, which allows organisms to adapt. Evidence suggests that many bacteria balance this trade-off by modulating their mutation rates, but few mechanisms have been described for such modulation. Following experimental evolution and whole-genome resequencing of the marine bacterium Vibrio splendidus 12B01, we discovered one such mechanism, which allows this bacterium to switch to an elevated mutation rate. This switch is driven by the excision of a mobile element residing in mutS, which encodes a DNA mismatch repair protein. When integrated within the bacterial genome, the mobile element provides independent promoter and translation start sequences for mutS-different from the bacterium's original mutS promoter region-which allow the bacterium to make a functional mutS gene product. Excision of this mobile element rejoins the mutS gene with host promoter and translation start sequences but leaves a 2-bp deletion in the mutS sequence, resulting in a frameshift and a hypermutator phenotype. We further identified hundreds of clinical and environmental bacteria across Betaproteobacteria and Gammaproteobacteria that possess putative mobile elements within the same amino acid motif in mutS In a subset of these bacteria, we detected excision of the element but not a frameshift mutation; the mobile elements leave an intact mutS coding sequence after excision. Our findings reveal a novel mechanism by which one bacterium alters its mutation rate and hint at a possible evolutionary role for mobile elements within mutS in other bacteria. IMPORTANCE: DNA mutations are a double-edged sword. Most mutations are harmful; they can scramble precise genetic sequences honed over thousands of generations. However, in rare cases, mutations also produce beneficial new traits that allow populations to adapt to changing environments. Recent evidence suggests that some bacteria balance this trade-off by altering their mutation rates to suit their environment. To date, however, we know of few mechanisms that allow bacteria to change their mutation rates. We describe one such mechanism, driven by the action of a mobile element, in the marine bacterium Vibrio splendidus 12B01. We also found similar mobile genetic sequences in the mutS genes of many different bacteria, including clinical and agricultural pathogens. These mobile elements might play an as yet unknown role in the evolution of these important bacteria.

Genetic and biochemical interactions between the bacterial replication initiator DnaA and the nucleoid-associated protein Rok in Bacillus subtilis.

We identified interactions between the conserved bacterial replication initiator and transcription factor DnaA and the nucleoid-associated protein Rok of Bacillus subtilis. DnaA binds directly to clusters of DnaA boxes at the origin of replication and elsewhere, including the promoters of several DnaA-regulated genes. Rok, an analog of H-NS from gamma-proteobacteria that affects chromosome architecture and of Lsr2 from Mycobacteria, binds A+T-rich sequences throughout the genome and represses expression of many genes. Using crosslinking and immunoprecipitation followed by deep sequencing (ChIP-seq), we found that DnaA was associated with eight previously identified regions containing clusters of DnaA boxes, plus 36 additional regions that were also bound by Rok. Association of DnaA with these additional regions appeared to be indirect as it was dependent on Rok and independent of the DNA-binding domain of DnaA. Gene expression and mutant analyses support a model in which DnaA and Rok cooperate to repress transcription of yxaJ, the yybNM operon and the sunA-bdbB operon. Our results indicate that DnaA modulates the activity of Rok. We postulate that this interaction might affect nucleoid architecture. Furthermore, DnaA might interact similarly with Rok analogues in other organisms.

Autonomous Replication of the Conjugative Transposon Tn916.

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916 was dependent on the relaxase encoded by orf20 of Tn916 The origin of transfer of Tn916, oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916 likely interact in a complex and that the Tn916 relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916 that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism. IMPORTANCE: Integrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.