First Report of Integrative Conjugative Elements in Riemerella anatipestifer Isolates From Ducks in China

We report for the first time the occurrence of integrative conjugative elements (ICEs) in Riemerella anatipestifer (R.anatipestifer) isolated from diseased ducks in China. For this purpose, a total of 48 genome sequences were investigated, which comprised 30 publicly available R. anatipestifer genome sequences, and 18 clinical isolates genomes sequences. Two ICEs, named ICERanRCAD0133-1 and ICERanRCAD0179-1 following the classic nomenclature system, were identified in R. anatipestifer through the use of bioinformatics tools. Comparative analysis revealed that three ICEs in Ornithobacterium rhinotracheale showed a high degree of conservation with the core genes of ICERanRCAD0133-1, while 13 ICEs with high similarity to ICERanRCAD0179-1 were found in Bacteroidetes. Based on the definition of ICE family, ICERanRCAD0179-1 was grouped in CTnDOT/ERL family; however, ICERanRCAD0133-1, which had no significant similarity with known ICEs, might be classified into a novel ICE family. The sequences of ICERanRCAD0133-1 and ICERanRCAD0179-1 were 70890 bp and 49166 bp in length, had 33.14 and 50.34% GC content, and contained 77 CDSs and 51 CDSs, respectively. Cargo genes carried by these two ICEs were predicted to encode: R-M systems, IS elements, a putative TonB-dependent receptor, a bacteriocin/lantibiotic efflux ABC transporter, a tetracycline resistance gene and more. In addition, phylogenetic analyses revealed that ICERanRCAD0179-1 and related ICEs were derived from a common ancestor, which may have undergone divergence prior to integartation into the host bacterial chromosome, and that the core genes co-evolved via a related evolutionary process or experienced only a low degree of recombination events during spread from a common CTnDOT/ERL family ancestor. Collectively, this study is the first identification and characterization of ICEs in R. anatipestifer; and provides new insights into the genetic diversity, evolution, adaptation, antimicrobial resistance, and virulence of R. anatipestifer

Excision, transfer, and integration of the bacteroides integrative and conjugative element CTnDOT

Increased resistances to antibiotics is a pressing problem in the world today. When new antibiotics are introduced, resistance to that antibiotic within the microbial community soon follows. One of the many contributing factors to increased resistances to antibiotics is the spread of mobile genetic elements that encode antibiotic resistance genes. These elements are able to transfer themselves to new host cells by conjugation, which may result in the widespread dissemination of an element throughout a given microbial population. The Bacteroides sp. are known for harboring wide array of integrative and conjugative elements. One of the most well studied examples is the 65kb CTnDOT, which encodes both erythromycin and tetracycline resistances. Bacteroides are prevalent within the human gastrointestinal tract, which means that CTnDOT is capable of transferring to other resident microbes within this environment. The transfer of CTnDOT is repressed under normal conditions, but when tetracycline is present the propagation of the element is stimulated through a complex regulatory cascade. The excision operon is a key component of this regulation, and is involved at many different levels of the regulatory cascade. As the name implies, the expression of the excision operon promotes the excision of CTnDOT from the host chromosome. Once the element is excised it needs somewhere to go, which the excision operon also facilitates by increasing the transcription of both the mobilization (mob) and transfer (tra) operons. These operons encode the proteins needed to assemble the mating apparatus, and the proteins responsible for shuttling the excised element to the mating apparatus for transfer. The work presented within this dissertation focuses on the different aspects of the excision operon. First the mechanistic properties of the excision proteins at the mob and tra region were investigated. It was shown that the Xis2d protein binds the DNA between the mob and tra promoters, and that the Exc protein can be recruited in the presence of Xis2d to help initiate the transcription of the mob operon. The contribution of each of the excision genes to the in vivo excision reaction was also analyzed. It was shown that each of the genes within the excision operon play a significant role in the excision reaction. The presence of xis2c, xis2d, and exc was essential for detecting excised product, which confirms that exc is required for in vivo excision. Also, a deletion of orf3 significantly reduced the production of excised products, which is the first time this gene has shown a discernable effect in a CTnDOT assay. This wasn't the only discovery of gene functionality that was uncovered, the previously uncharacterized orf2a and orf2b genes were found to contribute to the excision reaction. The excision, transfer, and mobilization of CTnDOT ultimately lead to propagation of this element into new recipients. Once within the recipient cell, the element integrates into an attB site located within the recipient chromosome. This dissertation also provides an in vivo analysis of CTnDOT integration, where 18 alternative attB sites were identified. In summation, the work presented within this dissertation has clarified the main aspects of CTnDOT propagation, by revealing important contributions of the excision operon to the regulation of the element

The ICE-flox Integrative Conjugative Element of Legionella pneumophila.

Legionella pneumophila is an accidental human pathogen that causes the bacterial pneumonia Legionnaire’s Disease. The bacteria are ubiquitous in freshwater environments and are spread by aerosolization of contaminated water from the built environment. Accessory traits carried on mobile genetic elements diversify the L. pneumophila and may contribute to persistence in stressful environments. One class of mobile elements are Integrative Conjugative Elements (ICEs) which encode cargo genes as well as type IV secretion system (T4SS) transfer apparatuses to direct their own transmission among a bacterial population. In this dissertation, I demonstrate that ICE-βox enhances L. pneumophila resistance to oxidative stresses encountered in vitro (such as bleach) and in macrophages. Specifically, this mobile element protects L. pneumophila from the toxic activities of the macrophage phagocyte oxidase. In addition to cargo genes predicted to repair oxidative damage, ICE-βox encodes a paralog of the master L. pneumophila life cycle regulator csrA. Bioinformatic analyses of 34 L. pneumophila ICE-associated T4SS reveals four families based on apparatus composition. Each T4SS family is genetically and phylogenetically linked with a distinct csrA paralog, suggesting functional interactions. Indeed, the ICE-βox csrA paralog csrT can repress ICE-βox traits as well as motility of the host bacterium. Finally, a preliminary epidemiologic survey identified ICE-βox in a majority of built environment L. pneumophila isolates. Accordingly, the hypothesis that chlorine-based disinfectants enrich for ICE-βox and increase L. pneumophila resilience and virulence warrants testing. By understanding the fitness advantages, regulation and prevalence of ICE-βox, disinfection protocols can be designed to eradicate persistent L. pneumophila and reduce its risk to humans.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113493/1/kjflynn_1.pd

Integrative and Conjugative Elements (ICEs): What They Do and How They Work

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

Transcriptional regulation of mobilization and transfer of the Bacteroides conjugative transposon CTnDOT

Bacteroides spp. are a primary inhabitant of the human colon, and play an important role in the health of the human host as Bacteroides are important for nutrient acquisition, the breakdown of starches that would otherwise go undigested, and the management of relevant pathogens such as C. difficile. Although generally considered a commensal, Bacteroides can become an opportunistic pathogen if it should escape the colon. Bacteroides is the most commonly isolated causative agent of anaerobic infections, and these infections are rather difficult to treat due to the prevalence of antibiotic resistance within this genus. The prevalence of resistance determinants in Bacteroides is often due to mobile genetic elements. One such element is a conjugative transposon (sometimes referred to as an integrative conjugative element, or ICE) called CTnDOT, which is a 65 kb ICE that encodes resistance to the antibiotics erythromycin and tetracycline. A notable feature of CTnDOT is that excision from the donor chromosome and conjugative transfer are coordinately regulated upon exposure of donor cells to tetracycline (Tc). While no transfer is detected in the absence of Tc, upon Tc induction a regulatory cascade ultimately stimulates synthesis of the excision proteins, which are required for excision of CTnDOT from the chromosome. These proteins also have a regulatory role, in that they are required for the transcriptional activation of the 13 kb tra operon that encodes the mating apparatus. The work presented in this dissertation has characterized a negative regulator, RteR, that appears to prevent conjugative transfer of CTnDOT in the absence of Tc by possibly initiating the formation of an intrinsic terminator within traB, thus truncating the transcript so there is no substrate for translation, and hence no proteins are formed to assemble the mating apparatus. For the first time, we also describe the regulation of the CTnDOT mobilization region. These three genes that encode the relaxases and coupling protein required for mobilization are organized in an approximately 4 kb operon that is regulated upon Tc induction. The excision proteins Xis2d and Exc are required for enhancement of mob transcription upon exposure to Tc. This differs from the neighboring divergently transcribed tra operon, which does not require Exc, but also requires Xis2d and in addition Xis2c. A negative regulator is preventing mob transcription in the absence of Tc, and we currently predict that a gene encoded downstream of intDOT, orf2, is the mob transcriptional repressor. Taken together, the work described in this dissertation has further shed light on the intricate regulation governing the conjugative transfer of CTnDOT

Legionella pneumophila Exploits the CsrA Superfamily to Adapt to a Range of Stresses.

Legionella pneumophila persists in incredibly diverse environments including intracellularly in myriad protists and mammilian lung macrophages, and extracellularly in natural and engineered water systems. Success in a given condition requires efficient regulation of appropriate gene and protein expression. A central regulator of the switch between two distinct expression profiles associated with the replicative and the transmissive cell types is CsrA, a highly conserved protein among many bacterial species. Interestingly, each of 14 surveyed strains in the Legionella genus encode between four and seven paralogs of the csrA gene. Every strain encodes a copy of the canonical csrA, as well as the non-redundant newly described csrR, which is a direct target of CsrA repression and itself promotes long-term survival in tap water. In addition, each strain encodes a variety of other csrA paralogs always associated with type IV secretion systems (T4SSs) in legionellae-specific horizontally acquired genomic islands referred to as integrative and conjugative elements (ICEs). Bioinformatic analyses suggest that specific sequences of csrA-like genes are co-inherited with specific lineages of T4SSs. Ectopic expression of one of these ICE-associated csrA paralogs, csrT, resulted in repression of both ICE-dependent and core genome-associated fitness traits, such as resistance to hydrogen peroxide and cellular infectivity, respectfully. The observation that the core genome csrR and the ICE-associated csrT both demonstrated regulatory effects on fitness suggests that the Legionella genus extensively utilizes the csrA superfamily to control expression of genes and proteins to expand environmental versatility.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113530/1/zackabb_1.pd

The role of Bacteroides Host Factor A in CTnDOT integration and Holliday junction resolution

Bacteroides are a common genus of Gram negative anaerobic bacteria within the human gut. Under normal conditions, Bacteroides spp. benefit the host by breaking down complex polysaccharides. However, when the gut is punctured, these species may escape to cause abscesses or infections within the blood. In the past, tetracycline was used as a treatment for these infections. However, Bacteroides opportunistic infections are now much more difficult and costly to treat due to the widespread presence of mobile genetic elements carrying antibiotic resistance genes. One of these mobile genetic elements, CTnDOT, moves by conjugation and site-selective recombination. It is an example of a Conjugative Transposon (CTn). These elements were later classified as Integrated Conjugative Elements (ICEs). They frequently carry resistance to antibiotics and always contain the genes to mediate their own transfer by conjugation. CTnDOT is 65 kb and carries genes for mobilization, transfer, integration, and excision in addition to resistance to tetracycline and erythromycin. Once integrated into the chromosome, ICEs such as CTnDOT are stably maintained. The integration reaction into the Bacteroides chromosome and the excision reaction necessary for transfer are catalyzed by an integrase, IntDOT. IntDOT is a member of the tyrosine recombinase family. It was previously known that a host factor was required for integration and that Escherichia coli Integration Host Factor (IHF) could substitute for the host factor in an in vitro integration assay. I purified DNA binding proteins from a Bacteroides thetaiotaomicron strain lacking known ICEs. The purified fractions were then tested in the in vitro integration assay. The active fraction contained a protein that we named BHFa for Bacteroides Host Factor A. Subsequent electrophoretic mobility shift assays and fluorescent footprinting assays revealed four BHFa binding sites within the attDOT DNA sequence of CTnDOT. Surprisingly, further experiments showed that other DNA bending proteins could effectively substitute for BHFa in the in vitro integration assay even when distantly related or entirely unrelated. The integration and excision reactions of CTnDOT and other tyrosine recombinases proceed through ordered strand exchanges. The first set of strand exchanges generates a Holliday junction (HJ) intermediate that isomerizes through an overlap region and which is resolved by a second set of strand exchanges. Most tyrosine recombinases require identical DNA sequences in the overlap region where the strand exchanges occur. However, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo. This ability implies a difference in the overall protein-DNA complex, called an intasome. In integration, the intasome involves at least two different proteins and two double stranded DNA molecules, attDOT and attB. For excision, the attL and attR DNA sites are required for the assembly of two separate excisive intasomes before being brought together to reform the circular element. As many as five proteins may participate in excision and four are required. In order to study the mechanisms of the integration and excision reaction, we constructed synthetic HJs. HJ intermediates were constructed by annealing four oligonucleotides from the different products and substrates. This creates the intermediate formed after the first set of strand exchanges. The synthetic HJs can be constructed with either identical or mismatched overlap regions. It had been previously shown that synthetic HJs containing only the IntDOT core sites could be processed to both products and substrates if the overlaps are identical in vitro. However, if the overlaps contain mismatches, the HJs are resolved back to substrates. This inability of IntDOT to process mismatched HJs to products may be due to the lack of arm-type sites (which IntDOT binds) and because other protein participants were absent. I hypothesized that BHFa (or another DNA-binding protein) and the arm-type sites are necessary to form the higher-ordered complexes called intasomes and to enable IntDOT to catalyze recombination through the mismatched bases. To test this, I have constructed larger synthetic HJs (composed of four annealed oligonucleotides) containing the arm-type sites. As with core-only HJs, they were resolved into either products or substrates when the overlap contains identical bases. I have also constructed core plus arm-type HJs with a mismatched overlap. When these HJs are incubated with both IntDOT and BHFa they are resolved to both substrates and products. Accordingly, it appears that both arm-types sites and BHFa are required to enable IntDOT to resolve a HJ with a mismatched overlap region

Investigation of the environmental effect on the Regulation and Transfer of Conjugative Transposon Tn916

Antibiotic resistance is a major global concern, as it has spread rapidly and prevents the treatment of infectious diseases. One of the main reasons for this is the acquisition of resistance genes carried on mobile genetic elements (MGEs). There are many different types of MGEs, including transposons, plasmids, insertion sequences, pathogenicity islands, bacteriophages and gene cassettes. Tn916 is a conjugative transposon that contains the tetracycline-resistance gene tet(M). This transposon transfers to a broad range of bacteria. In this study, the research aimed to determine the behaviour of Tn916 in the human oral cavity, which is the portal to the digestive system. Bacillus subtilis (BS34A) has been used as a Tn916 donor, and six Streptococcus spp. (S. oralis, S. pyogenes, S. salivarius, S. mutans, S. sanguinis and S. parasanguinis) and Enterococcus faecalis as recipients. The donor B. subtilis is considered as a transient bacterium within the oral cavity, normally inhabiting soil, and the recipients are all considered normal oral and gastrointestinal bacteria. Filter-mating assays were carried out in pairs between donor–recipients under conditions that mimic those found in the mouth. It was observed that B. subtilis was able to transfer Tn916 into S. oralis, S. pyogenes and E. faecalis under anaerobic conditions at frequencies ranging from 10‾⁹ to 10‾⁷ transconjugants per recipient within 1 min on solid and liquid media. In contrast, no transfer was observed aerobically or when mating with S. salivarius, S. mutans, S. sanguinis and S. parasanguinis as recipients. However, after a mutant strain of B. subtilis onTn916 (Δorf12 terminator of Tn916) was developed, transfer of Tn916 occurred from B. subtilis into S. oralis, S. pyogenes and E. faecalis aerobically at frequencies ranging from10‾⁹ to 10‾⁸ transconjugants per recipient and anaerobically only into S. pyogenes. Using qPCR, the copy number of circular Tn916 in different conditions (aerobic and anaerobic) with and without a recipient (E. faecalis JH2-2) was determined. There were10⁷copies/μl in the absence of a recipient in both conditions. However, in the presence of the recipient, the copy numbers increased significantly to 10⁸ copies/μl anaerobically compared to 10⁷ copies/μl aerobically. Furthermore, the role of the orf12 terminator in aerobic and anaerobic conditions was determined by quantifying the expression level using an enzymatic reporter assay. The transcription level from ptet(M) is controlled by the terminator on orf12, and it was almost the same in both conditions. However, the transcription level was significantly increased when orf12 terminator mutated and the condition was switched from aerobic to anaerobic. In conclusion, transient bacteria in the oral cavity may have ample opportunities to disseminate their DNA to the oral microbiota

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies

Regulation of the mobile genetic element ICEBs1 by a conserved repressor and anti-repressor

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010."February 2010." Cataloged from PDF version of thesis.Includes bibliographical references.The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-Phrl sensory system activate ICEBsl gene expression, excision, and transfer by inactivating the ICEBsl repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBslencoded anti-repressor ImmA (YdcM). Under ICEBsl-inducing conditions, ImmA cleaves ImmR at a specific site to induce the element. We found that changing the amount or the specific activity of ImmA can cause derepression of ICEBs1 without activation by RecA or RapI. We isolated and characterized mutations in immA (immAh) that cause derepression of ICEBsl gene expression in the absence of inducing signals. However, we also found that ImmA levels did not significantly change during activation by RapI, indicating that RapI-mediated induction is likely due to increased activity of ImmA. Therefore, we propose that RapI and RecA induce ICEBs1 by increasing its specific activity. Along with earlier observations, some ImmAh mutants highlighted the importance of ImmA's C-terminal sequence for regulation of ImmA protein levels. We demonstrated that GFP tagged with C-terminal residues of ImmA was less abundant in vivo than untagged GFP. We screened cells with mutations of ATP-dependent proteases for effects on ICEBsl expression, and found that ClpXP might play a role in regulating ImmA stability and ICEBs1 gene expression.(cont.) To learn more about the repressor, ImmR, we isolated and characterized mutants of immR (immR(ind-)) that attenuate induction of ICEBs1 gene expression under the normally inducing conditions of treatment with DNA damaging reagent and overproduction of RapI. All four identified immR(ind-) mutations fall within a stretch of 10 residues flanking the cleavage site, emphasizing the importance of this sequence for ImmR proteolysis and ICEBsl induction. To further characterize the C-terminal portion of ImmR, we demonstrated that it interacts with ImmA and with itself in yeast two-hybrid assays, indicating that this part of the protein likely functions in ImmR oligomerization and recognition of ImmR by ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mode of regulation by ImmA and ImmR may be conserved in various systems.by Baundauna Bose.Ph.D