Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

Abstract

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.histidine relaxase | antibiotic resistance | horizontal gene transfer | X-ray structure | Staphylococcus aureus A cquisition of exogenous genetic material by bacteria is achieved via conjugative DNA transfer of mobile genetic elements, such as plasmids and especially integrative and conjugative elements and integrative and mobilizable elements (1). Such processes of horizontal gene transfer (HGT) are considered a strong driving force in bacterial evolution and in the ability of bacteria to colonize different niches (2). In addition to permitting the rapid evolution of the bacterial pangenome, HGT is involved in the acquisition of genetic traits that may confer selective advantages to the recipient bacteria, including antibiotic resistance (3). This is particularly important when resistance genes encoded by mobile elements are spread explosively among bacteria in hospitals, posing a serious threat to public health systems (www.cdc.gov/drugresistance/threat-report-2013; www. who.int/drugresistance/documents/surveillancereport/en/). Thus, the so-called mobilome (4) participates in the spread of antibiotic resistance, which is expected to cause 10 million casualties annually by 2050, and the consequent huge economic burden (amr-review.org/sites/default/files/Report-52.15.pdf). This has generated a unanimous call for new approaches to deal with infectious diseases caused by pathogenic bacteria (5).A main performer in HGT is the protein relaxase, a topoisomeraselike enzyme that cleaves supercoiled plasmid DNA in a strandand sequence-specific manner and ligates it after cell-to-cell transfer. Relaxases start DNA transfer by conjugation on recognition of their target DNA, the origin of transfer (oriT), on which they mediate generation of a hairpin-loop structure that leaves the dinucleotide to be cleaved (the nic site) in a singlestranded (ss) DNA configuration (Fig. 1B) (6, 7). On the oriT, the relaxase assembles with other proteins p...

Complete labelling of pneumococcal DNA-binding proteins with seleno-L-methionine

“…Prediction of the secondary structure of MobMN199 ( Figure 1B) showed an alternant distribution of α-helices with β-strands, that is the α/β-fold, which is found in many of the HUH endonucleases (Chandler et al, 2013). This prediction was confirmed by circular dichroism analyses (Lorenzo-Díaz et al, 2011) and, later on, when we could solve the structure of MobMN199 (Pluta et al, 2017). Lorenzo-Díaz et al, 2011).…”

“…Unlabelled protein MobMN199 (Lorenzo-Díaz et al, 2011;Pluta et al, 2017;Studier and Moffatt, 1986) pET28relBE…”

“…The two above approaches require that the three-dimensional structure of the proteins is known to design the proper antimicrobials. In the case of S. pneumoniae and its mobile elements, not very many protein structures with a known function have been determined so far and they include plasmid-encoded proteins (Boer et al, 2009, Espinosa, 2013, Gomis-Ruth et al, 1998, Pluta et al, 2017. This lack of knowledge could be attributed to the research on pneumococcus being more focussed on its epidemiology and management than on finding novel drug targets.…”

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering

“…Looking more broadly at the Rep-DNA interactions in the context of the entire HUH-endonuclease superfamily, the sDBM is apparently a ubiquitous motif contributing to DNA recognition. This is illustrated by available relaxase structures captured in the pre-cleavage state 17,[28][29][30] . In Reps, the sDBM is located in the middle of the structure and consists of the fourth beta strand and a portion of the preceding loop.…”

“…However, while several structures of relaxase HUH-endonucleases in complex with their cognate DNA target sequences have been reported 17,[28][29][30] , there are no structures of viral Rep HUH-endonucleases in complex with ssDNA comprising the target sequence at the origin of replication ( ori ). Despite structurally superimposable active sites and a common overall core structure 31 , there are several structural elements of the larger relaxase proteins that do not exist in Reps, such as extensions of the C-terminus and internal loops with respect to Reps.…”