Nucleotide sequence and functional analysis of the tet (M)-carrying conjugative transposon Tn5251 of Streptococcus pneumoniae

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Streptococcus pyogenes Φ1207.3 Is a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D) and Capable of Lysogenizing Different Streptococci

Streptococcus pyogenes prophage phi 1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if phi 1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the phi 1207.3-carrying lysogen were treated with mitomycin C to assess if phi 1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if phi 1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of phi 1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 x 10(4) and 6.06 x 10(4) excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 x 10(3) and 7.68 x 10(3) phage particles per milliliter of supernatant. The phage preparations of phi 1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 x 10(-6) lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 10(3) phage particles. © 2023 Santoro et al

Computing Period and Shape of Oscillations in Piecewise Linear Lur'e Systems: a Complementarity Approach

International audienceAutonomous piecewise linear systems in the Lur'e form may exhibit periodic steady-state oscillations. For many practical systems belonging to this class the period and the shape of the oscillation is difficult to be predicted a priori. In this paper the complementarity approach is used to tackle the issue. The complementarity formalism is used to represent the closed-loop system and a phase condition acting as an anchor equation for the periodic solution. By discretizing the dynamics a mixed complementarity problem is formulated. The corresponding solution provides an accurate prediction of the steady-state oscillation and its period. Numerical results show the effectiveness of the proposed technique for the computation of stable and sliding periodic solutions. The analysis of the steady-state solution of a Colpitts oscillator is considered as an illustration

Excision and Circularization of Integrative Conjugative Element Tn5253 of Streptococcus pneumoniae

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

The Mobilome-Enriched Genome of the Competence-Deficient Streptococcus pneumoniae BM6001, the Original Host of Integrative Conjugative Element Tn5253, Is Phylogenetically Distinct from Historical Pneumococcal Genomes

Streptococcus pneumoniae is an important human pathogen causing both mild and severe diseases. In this work, we determined the complete genome sequence of the S. pneumoniae clinical isolate BM6001, which is the original host of the ICE Tn5253. The BM6001 genome is organized in one circular chromosome of 2,293,748 base pairs (bp) in length, with an average GC content of 39.54%; the genome harbors a type 19F capsule locus, two tandem copies of pspC, the comC1-comD1 alleles and the type I restriction modification system SpnIII. The BM6001 mobilome accounts for 15.54% (356,521 bp) of the whole genome and includes (i) the ICE Tn5253 composite; (ii) the novel IME Tn7089; (iii) the novel transposon Tn7090; (iv) 3 prophages and 2 satellite prophages; (v) 5 genomic islands (GIs); (vi) 72 insertion sequences (ISs); (vii) 69 RUPs; (viii) 153 BOX elements; and (ix) 31 SPRITEs. All MGEs, except for the GIs, produce excised circular forms and attB site restoration. Tn7089 is 9089 bp long and contains 11 ORFs, of which 6 were annotated and code for three functions: integration/excision, mobilization and adaptation. Tn7090 is 9053 bp in size, flanked by two copies of ISSpn7, and contains seven ORFs organized as a single transcriptional unit, with genes encoding for proteins likely involved in the uptake and binding of Mg2+ cations in the adhesion to host cells and intracellular survival. BM6001 GIs, except for GI-BM6001.4, are variants of the pneumococcal TIGR4 RD5 region of diversity, pathogenicity island PPI1, R6 Cluster 4 and PTS island. Overall, prophages and satellite prophages contain genes predicted to encode proteins involved in DNA replication and lysogeny, in addition to genes encoding phage structural proteins and lytic enzymes carried only by prophages. &amp; phi;BM6001.3 has a mosaic structure that shares sequences with prophages IPP69 and MM1 and disrupts the competent comGC/cglC gene after chromosomal integration. Treatment with mitomycin C results in a 10-fold increase in the frequency of &amp; phi;BM6001.3 excised forms and comGC/cglC coding sequence restoration but does not restore competence for genetic transformation. In addition, phylogenetic analysis showed that BM6001 clusters in a small lineage with five other historical strains, but it is distantly related to the lineage due to its unique mobilome, suggesting that BM6001 has progressively accumulated many MGEs while losing competence for genetic transformation