Dataset Information

Excision and Circularization of Integrative Conjugative Element Tn5253 of Streptococcus pneumoniae.

ABSTRACT: 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.

SUBMITTER: Santoro F

PROVIDER: S-EPMC6079316 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Excision and Circularization of Integrative Conjugative Element Tn5253 of Streptococcus pneumoniae.

Santoro Francesco F Romeo Alessandra A Pozzi Gianni G Iannelli Francesco F

Frontiers in microbiology 20180731

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 8 ...[more]

PMID: 30108581

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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.

Project description:Integrative conjugative elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation, and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules, including cell surface proteins, wall teichoic acids, lipoteichoic acids, and exopolysaccharides, on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild-type (WT) donor cells to mutated recipient cells increased 5- to 400-fold when recipient cells were affected in lipoproteins, teichoic acids, or exopolysaccharides compared to when the recipient cells were WT. These mutants displayed an increased biofilm-forming ability compared to the WT, suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation. In contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to that with the WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor cells suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport.IMPORTANCE ICEs contribute to horizontal gene transfer of adaptive traits (for example, virulence, antibiotic resistance, or biofilm formation) and play a considerable role in bacterial genome evolution, thus underlining the need of a better understanding of their conjugative mechanism of transfer. While most studies focus on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model, we demonstrated the impact of lipoproteins, teichoic acids, and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfer mediated by ICEs.

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.

Dataset Information

Excision and Circularization of Integrative Conjugative Element Tn5253 of Streptococcus pneumoniae.

Publications

Excision and Circularization of Integrative Conjugative Element Tn<i>5253</i> of <i>Streptococcus pneumoniae</i>.

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