Bacterial integrons are genetic platforms that play a vital role in the adaptability of Gram-negative bacteria, chiefly by enabling the acquisition and expression of gene cassettes that confer antibiotic resistance. This dynamic recombination process involves integrons capturing and excising gene cassettes through site-specific recombination, facilitated by an enzyme known as integrase. The integrase binds to single-stranded DNA segments called attC sites, folding the DNA into a hairpin structure and forms a synaptic complex. This complex facilitates site-specific recombination and integrates of gene cassettes into the bacterial genome, a key mechanism behind the adaptability and antibiotic resistance of bacteria.
The mystery of recombination efficiency of bacterial integrons
The recombination efficiency of bacterial integrons is critical as it directly affects how rapidly and effectively bacteria can respond to environmental pressures such as antibiotic exposure, which is central to their survival and proliferation in hostile environments. Interestingly, recombination efficiency shows remarkable variability, sometimes differing by up to five orders of magnitude, even when mediated by the same integrase enzyme across different attC sites. The underlying causes of these efficiency variations remain elusive, despite detailed understanding of the crystallographic structures of protein-protein and protein-DNA interactions within the integrase.
A recent study, by the team of Prof. Michael Schlierf from TU Dresden, employs C-Trap single-molecule optical tweezers technology, and finds a correlation between the mechanical stability of the synaptic complex and the efficiency of recombination, suggesting that mechanical properties could play a role in the efficiency of genetic exchange.
The experimental assay
The researchers constructed a hybrid DNA molecule by linking a single-stranded DNA (ssDNA) sequence containing two attC sites with double-stranded DNA (dsDNA) handles modified with biotin and triple-digoxigenin at the 5′ ends. Using the microfluidic multichannel chamber of the C-Trap, they first attached the hybrid DNA molecule between two beads which were trapped by optical tweezers, then they moved the molecule into the channel in the absence of integrase IntI1 and formed the synaptic complex. Later they moved one bead away from the other and reversed at a constant velocity. The force-extension curve was analyzed to determine the mechanical stability of the synaptic complex, with higher disassembly forces indicating greater stability.
Correlation between the structural stability and the recombination efficiency
This research depicted critical findings regarding the mechanical aspects of genomic recombination with bacterial integrons:
- A correlation exists between the mechanical stability of the synaptic complex and its recombination efficiency; complexes that demonstrated greater stability also exhibited higher recombination efficiencies.
- Variability in mechanical stability across different attC sites was observed, suggesting that structural differences at the genetic level influence overall complex stability.
- Alterations in integrase, through point mutations, affect not only protein-DNA binding affinity but also the broader mechanical stability of the synaptic complex, indicating a complicated role of integrase in genome recombination.
Scientific impact and future directions
This research, for the first time, establishes a link between the mechanical stability of synaptic complexes and their recombination efficiencies in vivo, unveiling a previously unknown mechanism that controls the stability of macromolecular complexes. This insight is pivotal for advancing our understanding of bacterial adaptation mechanisms, essential for developing strategies to combat antibiotic resistance. The significance of this finding highlights the importance of single-molecule biology in addressing not only fundamental biological functions and also global public health concerns.
Methodologically, the use of the C-Trap optical tweezers to measure the mechanical stability of synaptic complexes introduces a revolutionary approach in microbial genetics research. This technique enables precise, real-time observations of dynamic molecular processes, which may be applicable to other DNA-protein interaction systems. Given its potential in facilitating detailed mechanical measurements at the molecular level, we recommend all researchers in the field to consider adopting this technology.
Images are adapted from the original publication: Vorobevskaia E, Loot C, Mazel D, Schlierf M. The recombination efficiency of the bacterial integron depends on the mechanical stability of the synaptic complex. Science Advances. 2024 Dec 13;10(50):eadp8756.
