Determination of copy number and circularization ratio of Tn916-Tn1545 family of conjugative transposons in oral streptococci by droplet digital PCR.
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ABSTRACT:
SUBMITTER: Lunde TM
PROVIDER: S-EPMC6292373 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway.
| S-EPMC7971310 | biostudies-literature
Project description:The integrative conjugative element (ICE) Tn5253 of Streptococcus pneumoniae, conferring resistance to tetracycline and chloramphenicol, was found integrated at a 83-bp specific target site (attB) located in the rbgA gene of the pneumococcal chromosome. PCR analysis of Tn5253-carrying strains showed evidence of precise excision of Tn5253 from the pneumococcal chromosome with production of (i) circular forms of the ICE in which the ends were joined by a 84-bp sequence (attTn), and (ii) reconstituted chromosomal attB. When integrated into the chromosome, Tn5253 was flanked by attL, identical to attB, and attR, identical to attTn. Circular forms of Tn5253 were present at a concentration of 3.8 × 10-4 copies per chromosome, whereas reconstituted attB sites were at 3.0 × 10-4 copies per chromosome. Deletion of int-xis of Tn5253 abolished production of circular forms (<7.1 × 10-6 copies per chromosome) and was associated to the lack of Tn5253 conjugal transfer suggesting, as expected, that Tn5253 circular form acts as a conjugation intermediate.
| S-EPMC6079316 | biostudies-literature
Project description:Multidrug resistance in Streptococcus pneumoniae (or pneumococcus) continues to be a global challenge. An important class of antibiotic resistance determinants disseminating in S. pneumoniae are >20-kb Tn916-related integrative and conjugative elements (ICEs), such as Tn2009, Tn6002, and Tn2010. Although conjugation has been implicated as the transfer mechanism for ICEs in several bacteria, including S. pneumoniae, the molecular basis for widespread dissemination of pneumococcal Tn916-related ICEs remains to be fully elucidated. We found that Tn2009 acquisition was not detectable via in vitro transformation nor conjugative mating with donor GA16833, yielding a transfer frequency of <10-7. GA16833 Tn2009 conjugative gene expression was not significantly induced, and ICE circular intermediate formation was not detected in biofilms. Consistently, Tn2009 transfer efficiency in biofilms was not affected by deletion of the ICE conjugative gene ftsK. However, GA16833 Tn2009 transfer occurred efficiently at a recombination frequency (rF) of 10-4 in dual-strain biofilms formed in a human nasopharyngeal cell bioreactor. DNase I addition and deletions of the early competence gene comE or transformation apparatus genes comEA and comEC in the D39 recipient strain prevented Tn2009 acquisition (rF of <10-7). Genome sequencing and single nucleotide polymorphism analyses of independent recombinants of recipient genotype identified ~33- to ~55-kb donor DNAs containing intact Tn2009, supporting homologous recombination. Additional pneumococcal donor and recipient combinations were demonstrated to efficiently transfer Tn916-related ICEs at a rF of 10-4 in the biofilms. Tn916-related ICEs horizontally disseminate at high frequency in human nasopharyngeal S. pneumoniae biofilms by transformation and homologous recombination of >30-kb DNA fragments into the pneumococcal genome. IMPORTANCE The World Health Organization has designated Streptococcus pneumoniae as a priority pathogen for research and development of new drug treatments due to extensive multidrug resistance. Multiple strains of S. pneumoniae colonize and form mixed biofilms in the human nasopharynx, which could enable exchange of antibiotic resistance determinants. Tn916-related integrative and conjugative elements (ICEs) are largely responsible for the widespread presence of macrolide and tetracycline resistance in S. pneumoniae. Utilizing a system that simulates colonization of donor and recipient S. pneumoniae strains in the human nasopharynx, efficient transfer of Tn916-related ICEs occurred in human nasopharyngeal biofilms, in contrast to in vitro conditions of planktonic cells with exogenous DNA. This high-frequency Tn916-related ICE transfer between S. pneumoniae strains in biofilms was due to transformation and homologous recombination, not conjugation. Understanding the molecular mechanism for dissemination of Tn916-related ICEs can facilitate the design of new strategies to combat antibiotic resistance.
| S-EPMC10101023 | biostudies-literature
Project description:Enterobacterales are widely distributed in the gastro-intestinal system of animals and may cause opportunistic infections. Worse still, multidrug-resistant Enterobacterales also poses a serious threat to public health. Tn7-like transposons have been found in several species of the Enterobacterales order and play an important role in dissemination of antibiotic resistance. This study aimed to investigate the distribution and genetic characterization of Tn7-like transposons in Enterobacterales isolates from food animals and their association with antibiotic resistance. Enterobacterales isolated from the samples were identified and classified according to the 16S rDNA sequence. Tn7-like transposons and associated integrons were detected by polymerase chain reaction (PCR) and sequencing. The antibiotic resistance of each Tn7-like transposon positive isolate was detected according to the Kirby-Bauer disk diffusion method. Then, six representative strains were selected to study the genetic environment by whole-genome sequencing (WGS). In total, we isolated 377 Tn7-like transposons positive strains of Enterobacterales. Class 2 integrons were detected in 99.5% of the isolates, and there were high frequency mutation sites especially in base 535, a stop mutation. Many isolates (54.9%) were multidrug-resistant and observed high resistance rates to trimethoprim/sulfamethoxazole and streptomycin. Among these strains, we found three new types of Tn7-like transposons, named Tn6813, Tn6814, and Tn6765. This is the first comprehensive survey that shows Tn7-like transposons in Enterobacterales from animals used for food production in different regions of China. This study also provides an insight into the horizontal transfer of resistance genes associated with Tn7-like transposons.
| S-EPMC7500258 | biostudies-literature
Project description:While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.
| S-EPMC6914776 | biostudies-literature
Project description:Human colonic Bacteroides species harbor a family of large conjugative transposons, called tetracycline resistance (Tcr) elements. Activities of these elements are enhanced by pregrowth of bacteria in medium containing tetracycline, indicating that at least some Tcr element genes are regulated by tetracycline. Previously, we identified a central regulatory locus on the Tcr elements that contained two genes, rteA and rteB, which appeared to encode a two-component regulatory system (A. M. Stevens, J. M. Sanders, N. B. Shoemaker, and A. A. Salyers, J. Bacteriol. 174:2935-2942, 1992). In the present study, we describe a gene which is located downstream of rteB in a separate transcriptional unit and which requires RteB for expression. Sequence analysis of this gene showed that it encoded a 217-amino-acid protein, which had no significant sequence similarity to any proteins in the GenBank or EMBL data base. An insertional disruption in the gene abolished self-transfer of the Tcr element to Bacteroides recipients, indicating that the gene was essential for self-transfer. The disruption also affected mobilization of coresident plasmids. Mobilization frequency was reduced 100- to 1,000-fold if the recipient was Escherichia coli but was not affected to the same extent if the recipient was an isogenic Bacteroides strain. The complex phenotype of the disruption mutant suggested that the newly identified gene, like rteA and rteB, had a regulatory function. Accordingly, it has been designated rteC. Our results indicate that regulation of Tc(r) element functions is unexpectedly complex and may involve a cascade of regulators, with RteA and RteB exerting central control over secondary regulators like RteC, which in turn control subsets of Tcr element structural genes.
| S-EPMC206707 | biostudies-other
Project description:Fluorescence microscopy of cells expressing proteins translationally linked to a fluorophore can be a powerful tool to investigate protein localization dynamics in vivo. One major obstacle to reliably analyze biologically relevant localization is the construction of a fusion protein that is both fluorescent and functional. Here, we develop a strategy to construct fluorescent fusions at theoretically any location in the protein by using TnFLXopen random transposon mutagenesis to randomly insert a gene encoding a fluorescent protein. Moreover, insertions within a target gene are enriched by an inducible gene-trap strategy and selection by fluorescence activated cell sorting. Using this approach, we isolate a variety of fluorescent fusions to FtsZ that exhibit ring-like localization and a fusion to the flagellar stator protein that both is functional for supporting motility and localizes as fluorescent puncta. Finally, we further modify TnFLXopen to insert the coding sequence for the C-terminal half of mVenus for use in bimolecular fluorescence complementation (BiFC) and the in vivo detection of protein-protein interaction candidates. As proof-of-concept, the DivIVA polar scaffolding protein was fused to the N terminus of mVenus, the C terminus of mVenus was delivered by transposition, and a combination of fluorescence activated cell sorter (FACS) sorting and whole-genome sequencing identified the known self-interaction of DivIVA as well as other possible candidate interactors. We suggest that the FACS selection is a viable alternative to antibiotic selection in transposon mutagenesis that can generate new fluorescent tools for in vivo protein characterization. IMPORTANCE Transposon mutagenesis is a powerful tool for random mutagenesis, as insertion of a transposon and accompanying antibiotic resistance cassette often disrupt gene function. Here, we present a series of transposons with fluorescent protein genes which, when integrated in frame, may be selected with a fluorescence activated cell sorter (FACS). An open reading frame runs continuously through the transposon such that fluorescent protein fusions may be inserted theoretically anywhere in the primary sequence and potentially preserve function of the target protein. Finally, the transposons were further modified to randomly insert a partial fluorescent protein compatible with bimolecular fluorescence complementation (BiFC) to identify protein interaction candidates.
| S-EPMC9241775 | biostudies-literature
Project description:Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over thirty years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.
| S-SCDT-10_15252-EMBR_202357369 | biostudies-other
Project description:A total of 12 VanA-type vancomycin-resistant enterococci, consisting of 10 Enterococcus faecium isolates and two Enterococcus avium isolates, were examined in detail. The vancomycin resistance conjugative plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) were isolated from each of three different E. faecium strains. The plasmids transferred highly efficiently between enterococcus strains during broth mating and were homologous with pMG1 (Gm(r); 65.1 kb).
| S-EPMC262697 | biostudies-literature
Project description:Lateral gene transfer is a significant contributor to the ongoing evolution of many bacterial pathogens, including beta-hemolytic streptococci. Here we provide the first characterization of a novel integrative conjugative element (ICE), ICESde3396, from Streptococcus dysgalactiae subsp. equisimilis (group G streptococcus [GGS]), a bacterium commonly found in the throat and skin of humans. ICESde3396 is 64 kb in size and encodes 66 putative open reading frames. ICESde3396 shares 38 open reading frames with a putative ICE from Streptococcus agalactiae (group B streptococcus [GBS]), ICESa2603. In addition to genes involves in conjugal processes, ICESde3396 also carries genes predicted to be involved in virulence and resistance to various metals. A major feature of ICESde3396 differentiating it from ICESa2603 is the presence of an 18-kb internal recombinogenic region containing four unique gene clusters, which appear to have been acquired from streptococcal and nonstreptococcal bacterial species. The four clusters include two cadmium resistance operons, an arsenic resistance operon, and genes with orthologues in a group A streptococcus (GAS) prophage. Streptococci that naturally harbor ICESde3396 have increased resistance to cadmium and arsenate, indicating the functionality of genes present in the 18-kb recombinogenic region. By marking ICESde3396 with a kanamycin resistance gene, we demonstrate that the ICE is transferable to other GGS isolates as well as GBS and GAS. To investigate the presence of the ICE in clinical streptococcal isolates, we screened 69 isolates (30 GGS, 19 GBS, and 20 GAS isolates) for the presence of three separate regions of ICESde3396. Eleven isolates possessed all three regions, suggesting they harbored ICESde3396-like elements. Another four isolates possessed ICESa2603-like elements. We propose that ICESde3396 is a mobile genetic element that is capable of acquiring DNA from multiple bacterial sources and is a vehicle for dissemination of this DNA through the wider beta-hemolytic streptococcal population.
| S-EPMC2655516 | biostudies-literature