New Tool IMPRINT Enhances DNA Transformation in Various Bacterial Strains

With their unique capabilities, bacteria hold immense promise for improving various aspects of the world. From sustainable chemical production to crop protection and even fighting infections, genetically engineered bacteria offer exciting possibilities. However, a major bottleneck has existed – current methods only work effectively on a limited number of bacterial species.

Researchers at the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg, have made a significant breakthrough with a new method called IMPRINT. This innovative technique, published in Molecular Cell, utilizes cell-free systems to significantly enhance DNA transformation in a wider variety of bacterial strains.

Bacteria inhabit nearly every environment on Earth, including both the interior and exterior of the human body. Advancing our understanding of bacteria and their manipulation can lead to innovative methods for preventing, diagnosing, and treating infections. Additionally, harnessing bacteria's potential offers various societal benefits, such as creating sustainable biotechnological solutions for chemical production and protecting crops from disease, thereby reducing environmental impact.

Scientists must be able to change these bacteria's genetic makeup to realize these benefits. Nevertheless, the effective transformation of DNA - the introduction of foreign DNA into a cell - has long been a barrier to genetically modifying bacteria. This has restricted its use to a narrow range of microorganisms.

The existence of restriction-modification systems is a significant barrier. These defense mechanisms obliterate incoming foreign DNA that lacks the distinctive methylation pattern that marks the bacterial genome. To get past this barrier, several DNA methyltransferases and a strain-specific process are needed to add the bacterium's pattern to the DNA.

These enzymes bind methyl groups - small chemical groups made up of three hydrogen atoms and one carbon atom - to the bases of DNA strands. New strategies are required because the labor-intensive and difficult-to-scale replication or circumvention of these DNA methylation patterns can be achieved with current methods.

A team led by the Julius-Maximilians-Universität Würzburg (JMU) and the Helmholtz Institute for RNA-based Infection Research (HIRI), a site of the Braunschweig Helmholtz Centre for Infection Research (HZI), has developed a novel method to enhance DNA transformation and replicate such patterns to address this challenge.

Imitating Methylation Patterns Rapidly IN TXTL is what they call IMPRINT. As part of this technique, the researchers express a bacterium's unique set of DNA methyltransferases using a cell-free transcription-translation (TXTL) system, a liquid mixture that can generate ribonucleic acids (RNAs) and proteins from added DNA. The DNA is then delivered to the target bacterium after being methylated by the enzymes.

IMPRINT represents an entirely new use of TXTL. While TXTL is widely employed for various purposes, including producing hard-to-express proteins or as affordable diagnostic tools, it has not previously been utilized to overcome barriers to DNA transformation in bacteria.”

Chase Beisel, Head, RNA Synthetic Biology Department, Helmholtz Institute for RNA-based Infection Research

Beisel, who is also a Professor at the Julius-Maximilians-Universität Würzburg, spearheaded the study in collaboration with researchers from North Carolina State University (NC State) in Raleigh, USA.

Compared to existing methods, IMPRINT offers speed and simplicity.

Current approaches require either laboriously purifying individual DNA methyltransferases or expressing them in E. coli, which often proves cytotoxic. These methods can take days to weeks and only reconstitute a fraction of the bacterium's methylation pattern.”

Justin M. Vento, Study First Author, and Ph.D. Student, Department of Chemical and Biomolecular Engineering, North Carolina State University

The scientists showed that IMPRINT was capable of expressing a wide variety of DNA methyltransferases. Moreover, complex methylation patterns could be recreated by combining these enzymes. This significantly accelerated DNA transformation in bacteria, including the probiotic Bifidobacteria and the pathogen Salmonella, including a difficult-to-transform strain of the latter that has received less attention.

There are many possible uses in contemporary research and medicine. When it comes to bacteria that fight infections, like commensal bacteria or those that make antibacterial compounds, IMPRINT can enhance DNA transformation in clinical isolates of bacterial pathogens. These bacteria's genetic engineering may produce novel classes of antibiotics and cell-based treatments.

The research team aims to expand the use of IMPRINT. 

We want to make a wide variety of bacterial pathogens genetically tractable for research. Until now, certain bacteria have been favored as models simply because they are easier to genetically manipulate. We are hopeful that, by using IMPRINT, researchers will be able to focus on the most important bacterial strains, such as those with increased virulence or antibiotic resistance.”

Chase Beisel, Head, RNA Synthetic Biology Department, Helmholtz Institute for RNA-based Infection Research

He is optimistic that IMPRINT will gain widespread adoption within the research community.