Discovery of a new family of relaxases in Firmicutes bacteria

Abstract

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.

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] .…”

Analysis of COMPASS, a New Comprehensive Plasmid Database Revealed Prevalence of Multireplicon and Extensive Diversity of IncF Plasmids

“…Overall, we found that half of the plasmids (51%) of the COMPASS database are predicted to be mobilizable or self-transmissible. The percentage of mobile plasmid in our study is higher than the one (39%) found by Smillie et al (2010) suggesting that new relaxase and MPF sequences were identified in the last decade (Ramachandran et al, 2017).…”

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

“…The well-documented oriT 110 sequence [20], which is shown in Figure 1, predicted the transfer of the Bs and Spc resistance markers by BEST4305 and BEST4306, respectively, to the recipient BEST386. In the continuous mixing experiment, only the predicted transcipients appeared and increased in number with time ( Figure 4).…”

“…The pLS20 genes involved in CT activity were identified and categorized as type IV secretion system (T4SS) genes [18,19]. Molecular studies show that the unique CT kinetics involved in pLS20 transmission are regulated by growth-dependent expression of T4SS proteins [18][19][20].…”