Discovery of a new family of relaxases in Firmicutes bacteria.

Gayetri Ramachandran,D Roeland Boer,Isidro Crespo,Ling J Wu,Carlos Alfonso,Praveen K Singh,Juan Roman Luque-Ortega,Wilfried J J Meijer,César Gago-Córdoba,Jian An Hao,David Abia,Andrés Miguel-Arribas

doi:10.1371/journal.pgen.1006586

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.

Highlights

Horizontal gene transfer (HGT) processes are responsible for the spread of antibiotic resistance (AbR) which poses a serious economic and health problem worldwide
Genes conferring antibiotic resistance are often located on genetic elements that can be transferred to other bacteria
It is known that a large part of the gut microbiome corresponds to Grampositive bacteria, and many of these belong to the phylum Firmicutes

Summary

Introduction

Horizontal gene transfer (HGT) processes are responsible for the spread of antibiotic resistance (AbR) which poses a serious economic and health problem worldwide. Conjugation, which is the process by which a DNA element is actively transferred from a donor cell to a recipient cell through a dedicated transportation pore connecting the cells, is the main HGT route responsible for spreading AbR genes [1,2,3,4]. Firmicutes constitute a large fraction of the microbiota of fermented foods, many of which thrive in the gut, and for which it has been shown that they can harbor AbR genes [11]. Together, this illustrates the need to better understand the biology of plasmid-mediated conjugation in G+ bacteria in general, and in the phylum of Firmicutes in particular

Methods

Results

Discussion

Conclusion