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

Abstract

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 MOB T family that are related to rolling-circle replication proteins of the Rep_trans family. MOB T relaxases are encoded by ICEs of the ICE St3 /ICE Bs1 /Tn 916 superfamily, a superfamily widespread in Firmicutes, and frequently conferring antibiotic resistance. Results Here, we present the first biochemical and structural characterization of a MOB T relaxase: the RelSt3 relaxase encoded by ICE St3 from Streptococcus thermophilus . We identified the oriT region of ICE St3 and demonstrated that RelSt3 is required for its conjugative transfer. The purified RelSt3 protein is a stable dimer that provides a Mn 2+ -dependent single-stranded endonuclease activity. Sequence comparisons of MOB T relaxases led to the identification of MOB T 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 MOB T being a full family of non-canonical relaxases. The biochemical and structural characterization of a MOB T 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. Electronic supplementary material The online version of this article (10.1186/s13100-019-0160-9) contains supplementary material, which is available to authorized users.

Multiple plasmid origin-of-transfer substrates enable the spread of natural antimicrobial resistance to human pathogens

“…Multiple oriT regions were identified in over 63% of both the relaxase-typed as well as untyped mobile plasmids, where on average, 2 orits from 2 different Mob groups were identified per plasmid ( Fig 2G). This supported the notion that, besides secondary and in trans oriTs, the untyped plasmids likely carried un-identified relaxases 15,[20][21][22][23][24] . Moreover, the number of Fig 2H).…”

“…The standard approach for characterization of plasmid mobility involves classification of conjugation and mobilization genes 14 , especially typing of relaxase enzymes into the respective mobility groups 15,19 . However, besides the possibility of yet unidentified enzymes and mobility groups 15,[20][21][22][23][24] , multiple new processes have recently been uncovered that might confer additional mobility to plasmids and involve the origin-of-transfer (oriT) DNA substrate. These include (i) broadened relaxase binding specificities to multiple different oriT sequence variants [25][26][27][28][29] , which, according to the evolutionary theory of such DNA regions 4,30,31 , indicates the possibility of plasmids carrying multiple functional secondary oriTs, and (ii) trans-mobilization of plasmids carrying oriTs triggered by relaxases from co-resident plasmids acting in trans on the non-cognate oriTs [32][33][34][35] .…”

Abundance, Diversity and Role of ICEs and IMEs in the Adaptation of Streptococcus salivarius to the Environment

“…In this previous study [15], we also found that half of the streptococcal IME mobilization modules include a CP and that all these CPs except two do not belong to the canonical VirD4 superfamily but to the TcpA superfamily. Here, the percentage of S. salivarius IMEs/dIMEs encoding a CP is somewhat lower (33%, n = 54) and the majority of these CPs also belong to the TcpA superfamily, but the fraction is somewhat lower (77%, n = 42).…”

“…A 40% sequence identity clustering of CP allows to subdivide the TcpA superfamily into seven families, see Table 1, column 3. The TcpA_2, TcpA_6 and TcpA_12 families were already described [15], whereas TcpA_13, TcpA_14 and TcpA_15 are novel families. The most prevalent family is the TcpA_12 (n = 32), as previously described for streptococcal IMEs [16].…”

“…Three distinct superfamilies of relaxases have been identified thus far in ICEs from Firmicutes: the MobP, MobC and MobT [2,11,12]. The relaxase recognizes and cleaves one of the DNA strands of the circular element, specifically at the oriT site [13][14][15]. It is then recognized by a membrane-associated CP belonging to either the VirD4 or TcpA superfamilies.…”