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Abstract Background Two closely related ICEs, ICESt1 and ICESt3, have been identified in the lactic acid bacterium Streptococcus thermophilus. While their conjugation and recombination modules are almost identical (95% nucleotide identity) and their regulation modules related, previous work has demonstrated that transconjugants carrying ICESt3 were generated at rate exceeding by a 1000 factor that of ICESt1. Results The functional regulation of ICESt1 and ICESt3 transcription, excision and replication were investigated under different conditions (exponential growth or stationary phase, DNA damage by exposition to mitomycin C). Analysis revealed an identical transcriptional organization of their recombination and conjugation modules (long unique transcript) whereas the transcriptional organization of their regulation modules were found to be different (two operons in ICESt1 but only one in ICESt3) and to depend on the conditions (promoter specific of stationary phase in ICESt3). For both elements, stationary phase and DNA damage lead to the rise of transcript levels of the conjugation-recombination and regulation modules. Whatever the growth culture conditions, excision of ICESt1 was found to be lower than that of ICESt3, which is consistent with weaker transfer frequencies. Furthermore, for both elements, excision increases in stationary phase (8.9-fold for ICESt1 and 1.31-fold for ICESt3) and is strongly enhanced by DNA damage (38-fold for ICESt1 and 18-fold for ICESt3). Although ICEs are generally not described as replicative elements, the copy number of ICESt3 exhibited a sharp increase (9.6-fold) after mitomycin C exposure of its harboring strain CNRZ385. This result was not observed when ICESt3 was introduced in a strain deriving ICESt1 host strain CNRZ368, deleted for this element. This finding suggests an impact of the host cell on ICE behavior. Conclusions All together, these results suggest a novel mechanism of regulation shared by ICESt1, ICESt3 and closely related ICEs, which we identified by analysis of recently sequenced genomes of firmicutes. This is the first report of a partial shutdown of the activity of an ICE executed by a strain belonging to its primary host species. The sharp increase of ICESt3 copy number suggests an induction of replication; such conditional intracellular replication may be common among ICEs.

Conjugation is a key mechanism of bacterial evolution that involves mobile genetic elements. Recent findings indicated that the main actors of conjugative transfer are not the well-known conjugative or mobilizable plasmids but are the integrated elements. This paper reviews current knowledge on "integrative and mobilizable elements" (IMEs) that have recently been shown to be highly diverse and highly widespread but are still rarely described. IMEs encode their own excision and integration and use the conjugation machinery of unrelated co-resident conjugative element for their own transfer. Recent studies revealed a much more complex and much more diverse lifecycle than initially thought. Besides their main transmission as integrated elements, IMEs probably use plasmid-like strategies to ensure their maintenance after excision. Their interaction with conjugative elements reveals not only harmless hitchhikers but also hunters that use conjugative elements as target for their integration or harmful parasites that subvert the conjugative apparatus of incoming elements to invade cells that harbor them. IMEs carry genes conferring various functions, such as resistance to antibiotics, that can enhance the fitness of their hosts and that contribute to their maintenance in bacterial populations. Taken as a whole, IMEs are probably major contributors to bacterial evolution. [ABSTRACT FROM AUTHOR]

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by wholegenome sequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3= end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appear particularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcus faecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase. The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restrictionmodification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius. Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating that diffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present. [ABSTRACT FROM AUTHOR]

Examines the insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae. Recombination of genome evolution; Trapping of functional heterologous genes; Lack of correlation between concomitant chromosomal deletions and sizes of foreign DNA insertions.

Streptococcus suis is a zoonotic pathogen causing important economic losses in swine production. The most commonly used antibiotics in swine industry are tetracyclines, beta-lactams, and macrolides. Resistance to these antibiotics has already been observed worldwide (reaching high rates for macrolides and tetracyclines) as well as resistance to aminoglycosides, fluoroquinolones, amphenicols, and glycopeptides. Most of the resistance mechanisms are encoded by antibiotic resistance genes, and a large part are carried by mobile genetic elements (MGEs) that can be transferred through horizontal gene transfer. This review provides an update of the resistance genes, their combination in multidrug isolates, and their localization on MGEs in S. suis. It also includes an overview of the contribution of biofilm to antimicrobial resistance in this bacterial species. The identification of resistance genes and study of their localization in S. suis as well as the environmental factors that can modulate their dissemination appear essential in order to decipher the role of this bacterium as a reservoir of antibiotic genes for other species. [ABSTRACT FROM AUTHOR]

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes. [ABSTRACT FROM AUTHOR]

Streptococcus salivarius is a significant contributor to the human oral, pharyngeal and gut microbiomes that contribute to the maintenance of health. The high genomic diversity observed in this species is mainly caused by horizontal gene transfer. This work aimed to evaluate the contribution of integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) in S. salivarius genome diversity. For this purpose, we performed an in-depth analysis of 75 genomes of S. salivarius and searched for signature genes of conjugative and mobilizable elements. This analysis led to the retrieval of 69 ICEs, 165 IMEs and many decayed elements showing their high prevalence in S. salivarius genomes. The identification of almost all ICE and IME boundaries allowed the identification of the genes in which these elements are inserted. Furthermore, the exhaustive analysis of the adaptation genes carried by these elements showed that they encode numerous functions such as resistance to stress, to antibiotics or to toxic compounds, and numerous enzymes involved in diverse cellular metabolic pathways. These data support the idea that not only ICEs but also IMEs and decayed elements play an important role in S. salivarius adaptation to the environment. [ABSTRACT FROM AUTHOR]

Streptococcus suis is a zoonotic pathogen suspected to be a reservoir of antimicrobial resistance (AMR) genes. The genomes of 214 strains of 27 serotypes were screened for AMR genes and chromosomal Mobile Genetic Elements (MGEs), in particular Integrative Conjugative Elements (ICEs) and Integrative Mobilizable Elements (IMEs). The functionality of two ICEs that host IMEs carrying AMR genes was investigated by excision tests and conjugation experiments. In silico search revealed 416 ICE-related and 457 IME-related elements. These MGEs exhibit an impressive diversity and plasticity with tandem accretions, integration of ICEs or IMEs inside ICEs and recombination between the elements. All of the detected 393 AMR genes are carried by MGEs. As previously described, ICEs are major vehicles of AMR genes in S. suis. Tn5252-related ICEs also appear to carry bacteriocin clusters. Furthermore, whereas the association of IME-AMR genes has never been described in S. suis, we found that most AMR genes are actually carried by IMEs. The autonomous transfer of an ICE to another bacterial species (Streptococcus thermophilus)—leading to the cis-mobilization of an IME carrying tet(O)—was obtained. These results show that besides ICEs, IMEs likely play a major role in the dissemination of AMR genes in S. suis. [ABSTRACT FROM AUTHOR]

Background: Conjugative spread of antibiotic resistance and virulence genes in bacteria constitutes an important threat to public health. Beyond the well-known conjugative plasmids, recent genome analyses have shown that integrative and conjugative elements (ICEs) are the most widespread conjugative elements, even if their transfer mechanism has been little studied until now. The initiator of conjugation is the relaxase, a protein catalyzing a site-specific nick on the origin of transfer (oriT) of the ICE. Besides canonical relaxases, recent studies revealed non-canonical ones, such as relaxases of the MOBT family that are related to rolling-circle replication proteins of the Rep_trans family. MOBT relaxases are encoded by ICEs of the ICESt3/ICEBs1/Tn916 superfamily, a superfamily widespread in Firmicutes, and frequently conferring antibiotic resistance. Results: Here, we present the first biochemical and structural characterization of a MOBT relaxase: the RelSt3 relaxase encoded by ICESt3 from Streptococcus thermophilus. We identified the oriT region of ICESt3 and demonstrated that RelSt3 is required for its conjugative transfer. The purified RelSt3 protein is a stable dimer that provides a Mn2+-dependent single-stranded endonuclease activity. Sequence comparisons of MOBT relaxases led to the identification of MOBT conserved motifs. These motifs, together with the construction of a 3D model of the relaxase domain of RelSt3, allowed us to determine conserved residues of the RelSt3 active site. The involvement of these residues in DNA nicking activity was demonstrated by targeted mutagenesis. Conclusions: All together, this work argues in favor of MOBT being a full family of non-canonical relaxases. The biochemical and structural characterization of a MOBT member provides new insights on the molecular mechanism of conjugative transfer mediated by ICEs in Gram-positive bacteria. This could be a first step towards conceiving rational strategies to control gene transfer in these bacteria. [ABSTRACT FROM AUTHOR]

The diversity of clinical (n=92) and oral and digestive commensal (n=120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/ E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide- lincosamide-streptogramin B [MLSB] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n=13), followed by Tn6002 (n=11) and Tn2009 (n=4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut. [ABSTRACT FROM AUTHOR]