About AMR: Uncovering enzyme structure sheds light on transfer mechanisms for antibiotic resistant genes

Horizontal gene transfer plagues the control of multi-drug resistant infections, but how exactly are genes transferred and can we stop them? In a recent Cell paper, Rubio-Cosials and colleagues detail the biochemical events leading up to the transfer of transposons carrying antibiotic resistance genes. Their study uncovers important features of a key enzyme involved in DNA transfer and supports the rational design of drugs that could limit the spread of antibiotic resistance genes.

Transposons are DNA segments that move from one place in a genome to another through a series of events catalyzed by an element-encoded enzyme called transposase. Transposases are a broad group of enzymes that all share the ability to cleave and recombine DNA, but can be categorized into smaller groups based on the chemistry they use to catalyze DNA recombination. These differences are dictated by the content and spatial arrangement of the amino acids that reside in the active site of the enzyme.

Whereas the biochemical details of certain transposase groups are well characterized, others remain poorly understood, leaving gaps in our understanding of the basic chemical reactions that allow transposases to move DNA.

Tyrosine-transposases are a group of transposases whose biochemistry has remained elusive. These transposases move conjugative transposable elements – a unique subset of transposons that circularize following excision to generate an intermediate that can be transferred between bacteria. As such, conjugative transposons are a major source of genetic traits that are acquired by bacteria and equip them with new functions, such as antibiotic resistance. Indeed, conjugative transposons are responsible for a large proportion of antibiotic resistance in different pathogens, such as vancomycin, tetracycline, kanamycin, and macrolide resistance.

Unique to tyrosine-transposases is their ability to integrate DNA at seemingly random locations in the target genome. This level of promiscuity is not observed in other transposases and suggests an unusual enzyme chemistry. In a recent study published in Cell, Rubio-Cosials and colleagues revealed the structure of the tyrosine-transposase in complex with DNA, uncovering the features of the enzyme that permit its unique chemistry and shedding light on how conjugative transposons, and therefore antibiotic resistance, are spread.

The structure of the enzyme revealed several regions of the protein that are essential to its function. The first is a region exclusive to tyrosine-transposases that interacts with the DNA adjacent to the transposon sequence that inserts into the target DNA. This interaction distorts the DNA conformation to promote the initial cleavage of the transposon DNA. The nicked base then needs to find the new strand of DNA where it will insert. The researchers identified an amino acid residue in the enzyme that flips out the nicked base to nudge it into proximity of the target strand. Without this nudge, the enzyme is less efficient at strand exchange and integration.

The researchers then asked whether any DNA base could initiate strand exchange. By swapping the DNA bases in the transposon sequence, the researchers determined that a thymine base is required for strand exchange. This finding explains previous observations that conjugative transposons are generally associated with AT-rich sites, but are otherwise randomly interspersed throughout genomes.

The transposase enzyme must assemble as a dimer on the DNA strand to function properly. When the researchers compared various transposase sequences they noted a highly conserved region that, upon mapping to their structural data, they hypothesized would mediate protein-protein interactions. Indeed, when they removed the conserved region, the protein did not assemble properly on the DNA. Interestingly, enzyme activity increased. The researchers then mimicked this auto-inhibitory region with a small molecule to demonstrate that they could reduce enzyme activity and DNA recombination.

Altogether, this study reveals structural features in the tyrosine-transposase that are required for its unique transposase activity and demonstrate that this information can be exploited for the rational design of novel DNA recombination inhibitors. Such inhibitors could be used to limit the spread of antibiotic resistance.

Source: Rubio-Cosials A, Schulz EC, Lambertsen L, Smyshlyaev G, et al., Transposase-DNA complex structures reveal mechanisms for conjugative transposition of antibiotic resistance. Cell 173(1), 208–220 (2018)

Read more of Julie’s columns:

About the author

Julie Kaiser is a Ph.D. candidate in Microbiology at the University of Western Ontario in London, Canada. Her research interests range from the impact of microbial communities on human health, to the molecular basis of drug-resistant bacterial infections. Julie enjoys sharing her love for the microbial world through science communication and is the author of Microbiology for Dummies. Follow her on twitter at @jukais.


Leave A Comment