Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus in Physiological Conditions Mimicking the Human Digestive Ecosystem.

Metagenome analyses of the human microbiome suggest that horizontal gene transfer (HGT) is frequent in these rich and complex microbial communities. However, so far, only a few HGT studies have been conducted in vivo. In this work, three different systems mimicking the physiological conditions encountered in the human digestive tract were tested, including (i) the TNO gastro-Intestinal tract Model 1 (TIM-1) system (for the upper part of the intestine), (ii) the ARtificial COLon (ARCOL) system (to mimic the colon), and (iii) a mouse model. To increase the likelihood of transfer by conjugation of the integrative and conjugative element studied in the artificial digestive systems, bacteria were entrapped in alginate, agar, and chitosan beads before being placed in the different gut compartments. The number of transconjugants detected decreased, while the complexity of the ecosystem increased (many clones in TIM-1 but only one clone in ARCOL). No clone was obtained in a natural digestive environment (germfree mouse model). In the human gut, the richness and diversity of the bacterial community would offer more opportunities for HGT events to occur. In addition, several factors (SOS-inducing agents, microbiota-derived factors) that potentially increase in vivo HGT efficiency were not tested here. Even if HGT events are rare, expansion of the transconjugant clones can happen if ecological success is fostered by selecting conditions or by events that destabilize the microbial community. IMPORTANCE The human gut microbiota plays a key role in maintaining normal host physiology and health, but its homeostasis is fragile. During their transit in the gastrointestinal tract, bacteria conveyed by food can exchange genes with resident bacteria. New traits acquired by HGT (e.g., new catabolic properties, bacteriocins, antibiotic resistance) can impact the gut microbial composition and metabolic potential. We showed here that TIM-1, a system mimicking the upper digestive tract, is a useful tool to evaluate HGT events in conditions closer to the physiological ones. Another important fact pointed out in this work is that Enterococcus faecalis is a good candidate for foreign gene acquisition. Due to its high ability to colonize the gut and acquire mobile genetic elements, this commensal bacterium could serve as an intermediate for HGT in the human gut.

Chromosomal Conjugative and Mobilizable Elements in Streptococcus suis: Major Actors in the Spreading of Antimicrobial Resistance and Bacteriocin Synthesis Genes.

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.

Characterization of a relaxase belonging to the MOBT family, a widespread family in Firmicutes mediating the transfer of ICEs.

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.

Using molecular tools to decipher the complex world of plant resistance inducers: an apple case study.

Exogenous application of plant resistance inducers (PRIs) able to activate plant defenses is an interesting approach for new integrated pest management practices. The full integration of PRIs into agricultural practices requires methods for the fast and objective upstream screening of efficient PRIs and optimization of their application. To select active PRIs, we used a molecular tool as an alternative to methods involving plant protection assays. The expressions of 28 genes involved in complementary plant defense mechanisms were simultaneously determined by quantitative real-time PCR in PRI-treated tissues. Using a set of 10 commercial preparations and considering the pathosystem apple/Erwinia amylovora, this study shows a strong correlation between defense activation and protection efficiency in controlled conditions, thus enabling the easy identification of promising PRIs in fire blight protection. Hence this work clearly highlights the benefits of using a molecular tool to discriminate nonactive PRI preparations and provides useful molecular markers for the optimization of their use in orchard.