Abstract
Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.
Highlights
Conjugation constitutes one of the major mechanisms of horizontal gene transfer (Frost et al, 2005)
A great effort was made to understand the mode of action of conjugative type IV secretion systems (Conj-T4SSs) due to their major role in antibiotic resistance spreading among bacteria
Multiple structural information has been gained for individual components/proteins and isolated complexes improving our understanding of their architecture and assembly mode (reviewed in (Grohmann et al, 2018))
Summary
Conjugation constitutes one of the major mechanisms of horizontal gene transfer (Frost et al, 2005). VirB4 and VirB11 belong to traffic ATPases and are involved in the functioning of T4SS apparatus and in pili biogenesis whereas the coupling protein VirD4 (TC4P) is involved in DNA substrate recognition. The third class forms the outer membrane core complex It is composed of VirB7, VirB9, and VirB10 proteins. Other proteins are involved in the conjugative process These proteins include the transglycosylase VirB1 that is important for the establishment of the T4SS apparatus within the peptidoglycan layer in the periplasmic space and of the extracellular pili involved in DNA transfer or in host cell attachment. Initial steps of DNA recruitment are conducted by the relaxosome complex constituted by the relaxase and accessory factors that recognise and bind to the origin of transfer oriT sequence located in the conjugative element. Once transferred to the recipient cell, relaxase catalyses the recircularization of the T-strand followed by the second strand synthesis (Ilangovan et al, 2017)
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