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Transposable elements in prokaryotes are found in many forms and therefore a robust nomenclature system is needed in order to allow researchers to describe and search for them in publications and databases. Here we provide an update on The Transposon Registry which allocates numbers to any prokaryotic transposable element. Additionally, we present the completion of registry records for all transposons assigned Tn numbers from Tn 1 onwards where sequence data or publications exist.

Background Clostridium difficile strain 630Δ erm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns. Results In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn 5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain. Conclusions Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile .

Background Tn 5253 , a composite Integrative Conjugative Element (ICE) of Streptococcus pneumoniae carrying tet (M) and cat resistance determinants, was found to (i) integrate at specific 83-bp integration site ( att B), (ii) produce circular forms joined by a 84-bp sequence ( att Tn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the att B in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn 5253 att B site into bacterial genomes. Results Analysis of representative Tn 5253 -carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae , Streptococcus gordonii , Streptococcus pyogenes , and Enterococcus faecalis showed that: (i) Tn 5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the att B site corresponds to length of the duplication after Tn 5253 integration, (iv) att B duplication restores rbgA CDS, (v) Tn 5253 produced circular forms containing the att Tn site at a concentration ranging between 2.0 × 10 −5 to 1.2 × 10 −2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of att B sites occurred at 3.7 × 10 −5 to 1.7 × 10 −2 copies per chromosome. A database search of complete microbial genomes using Tn 5253 att B as a probe showed that (i) thirteen att B variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the att B site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species att B was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of att B sites, is conserved among the different bacterial species and can be considered the core of the Tn 5253 integration site. Conclusions A functional characterization of the Tn 5253 att B integration site combined with genome analysis contributed to elucidating the potential of Tn 5253 horizontal gene transfer among different bacterial species.

Abstract Broad host range conjugative plasmids that replicate in Escherichia coli have been widely used to mobilise smaller replicons, bearing their cognate origin of transfer (oriT) into a variety of organisms that are less tractable genetically, such as Clostridium (Clostridioides) difficile. In this work we demonstrated that the oriT region of pMTL9301 (derived from RK2) is not required for transfer between E. coli and C. difficile strains 630Δerm and CD37 and that this oriT-independent transfer is abolished in the presence of DNase when CD37 is the recipient. Transfer to the 630Δerm strain is DNase resistant even without an obvious oriT, when E. coli CA434 is used as a donor and is sensitive to DNase when E. coli HB101 is the donor. Clostridium difficile can acquire DNA from its environment, allowing it to rapidly respond to environmental change.

Bacillus subtilis strains BS49 and BS34A, both derived from a common ancestor, carry one or more copies of Tn916, an extremely common mobile genetic element capable of transfer to and from a broad range of microorganisms. Here, we report the complete genome sequence of BS49 and the draft genome sequence of BS34A, which have repeatedly been used as donors to transfer Tn916, Tn916 derivatives or oriTTn916-containing plasmids to clinically important pathogens. The genomes of two Bacillus subtilis strains with a common mobile element (Tn916) have been sequenced.

The human oral cavity is one of the hotspots harboring multiple mobile genetic elements (MGEs), which are segments of DNA that can move either within bacterial genomes or between bacterial cells that can facilitate the spreading of genetic materials, including antimicrobial resistance genes. It is, therefore, important to investigate genes associated with the MGEs as they have a high probability of dissemination within the bacterial population under selective pressure from human activities. As one-third of oral bacteria are not yet culturable in the laboratory condition, therefore, in this work, it is aimed to detect and identify the genetic contexts of MGEs in the oral cavity through an inverse PCR (IPCR)-based approach on the oral metagenomic. The human oral metagenome was extracted from saliva samples collected from healthy individuals in Tromsø, Norway. The extracted DNA was partially digested with the HindIII restriction enzyme and self-circularized by ligation. DNA primers targeting each MGE were designed to amplify outwards from the MGEs and used for the IPCR on the circularized DNA products. The IPCR amplicons were cloned into a pCR-XL-2-TOP vector, screened, and sequenced. Out of 40 IPCR amplicons, we confirmed and verified the genetic contexts of 11 samples amplified with primers targeting integron gene cassettes (GCs), IS 431 composite transposons, and Tn 916 conjugative transposons ( tet (M) and xis - int ). Novel integron GCs, MGEs, and variants of Tn 916 conjugative transposons were identified, which is the first report using the IPCR technique to detect the genetic contexts of MGEs in the oral metagenomic DNA.

Background Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius . Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046. Results JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA -type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM . Conclusion Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.

The conjugative transposon Tn 916 was determined to be functional in Paenibacillus larvae in regard to expression of tetracycline resistance and conjugative transfer. Expression of erythromycin resistance, using Tn 916 ΔE, was also observed. Conjugative transfer experiments employing Paenibacillus popilliae strains Tc1001 and Em1001 as transposon donors and experiments using different P. larvae subspecies or different transposon-containing strains demonstrated interspecies and intraspecies transfer occurred for Tn 916 and Tn 916 ΔE. Southern hybridization analysis of several Tn 916 -containing P. larvae isolates showed that the transposon randomly inserted into the bacterial chromosome with an indication that hot spot insertion had occurred. Hybridization analysis indicated single-copy insertion of Tn 916 into the genome predominated. However, selection of multiple-resistant isolates (i.e., isolates containing Tn 916 and Tn 916 ΔE) demonstrated that multiple copies of the transposon could coexist in the bacterial genome. Growth of transposon-containing isolates in broth medium in the absence of selective antibiotic pressure showed that Tn 916 and Tn 916 ΔE were stably maintained in the bacterium.

The Tn916-like genetic element Tn5251 is part of the composite conjugative transposon (CTn) Tn5253 of Streptococcus pneumoniae, a 64.5-kb chromosomal element originally called Ω(cat-tet) BM6001. DNA sequence analysis showed that Tn5251 is 18 033-bp long and contains 22 ORFs, 20 of which have the same direction of transcription. Annotation was possible for 11 out of 22 ORFs, including the tet(M) tetracycline resistance gene and int and xis involved in the integration/excision process. Autonomous copies of Tn5251 were generated during matings of Tn5253-containing donors with S. pneumoniae and Enterococcus faecalis. Tn5251 was shown to integrate at different sites in the bacterial chromosome. It behaves as a fully functional CTn capable of independent conjugal transfer to a variety of bacterial species including S. pneumoniae, Streptococcus gordonii, Streptococcus pyogenes, Streptococcus agalactiae, E. faecalis and Bacillus subtilis. The excision of Tn5251 produces a circular intermediate and a deletion in Tn5253 at a level of 1.2 copies per 10⁵ chromosomes.