2022 Lister Fellow Tung Le is Group Leader and Professor at John Innes Centre in Norwich, where he and his team explore mechanisms of genetic transfer in bacteria.
Working with an international team of microbiologists hailing from the UK, the US and Spain, Tung and his group recently made a significant discovery about an important plasmid – a small DNA molecule separate from the chromosome. This breakthrough solved a long-standing biological mystery that first captured Tung’s curiosity as an undergraduate.
We caught up with Tung to learn more about the discovery and the work that went into the resulting research paper, KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid
Q: Can you start by telling us about the plasmid you’ve been studying?
A: The plasmid is called RK2, and it was first discovered in a Pseudomonas bacterium about 40 years ago. It was originally isolated from a hospital patient in Birmingham. What makes it significant is that it confers multi-drug resistance and has a very broad host range. RK2 isn’t just found in Pseudomonas but in several other Gram-negative bacteria as well. Since it has the ability to transfer drug resistance among different pathogens, it has been extensively studied over the years.
Q: What specific aspect of RK2 did your research focus on?
A: We focused on understanding two proteins encoded within RK2 called KorA and KorB, and how they control expression of genes from this plasmid. The regulation of gene expression is a crucial biological process. Living organisms precisely control transcription to ensure the timely production of necessary products in the right amounts.
Previous studies had shown that KorA and KorB are essential for the survival of this plasmid. If you remove them, not only does the plasmid fail to propagate, but the host bacterium also dies. Despite many studies, there was no clear model explaining how KorA and KorB function together to regulate expression of genes on RK2. Some researchers thought KorB formed a DNA loop, while others proposed that they worked through polymerisation, to reach the target genes, thereby controlling their gene expression. Our goal was to resolve these conflicting models and uncover the true mechanism at play.
[As a student] I had assumed that everything in textbooks was already fully understood, but realising that there were still mysteries left to solve was exciting.
Q: You’ve been interested in these proteins for quite some time. What sparked your curiosity?
A: I first learned about KorA and KorB as an undergraduate at the University of Birmingham between 2004 and 2007. My lecturer, Prof. Chris Thomas – who is now a co-author on our paper – taught us about the RK2 plasmid. I remember him saying gene expression regulation of RK2 is fascinating, and we don’t yet fully understand how KorA and KorB actually work together to control long-range gene expression. That moment really stuck with me. I had assumed that everything in textbooks was already fully understood, but realising that there were still mysteries left to solve was exciting.
Q: How did you end up leading the team that finally solved the mystery?
A: About four years ago, a major development occurred in the field. Two groups discovered that ParB, which is crucial for bacterial chromosome segregation (and KorB is a member of this family of proteins), can bind a small molecule called CTP to form a sliding clamp around DNA. That discovery reignited my curiosity about KorA and KorB. I started thinking about Chris Thomas’s lectures again and realised it was the perfect time to revisit all the conflicting models. As we investigated, things became even more intriguing. Eventually, we were able to resolve the discrepancies and demonstrate that KorB functions as a sliding clamp on DNA, while KorA acts as a stopper, ensuring KorB arrives at the right target gene to regulate gene expression.
Q: That must have been a very satisfying discovery!
A: Absolutely! While sliding clamp proteins have been studied in recent years, it was rare that a partner protein was found to trap a sliding clamp at a specific location. It’s an elegant biological mechanism.
The discovery was incredible for me, tying together something I had learned as an undergraduate with my current research. My time at the University of Birmingham was pivotal in my career – it provided me with a full scholarship as an international student, which was rare at the time. The education I received there has been fundamental to all of my current work.
It was also particularly rewarding for Tom McLean, who played a crucial role in the investigation. While I was initially focused on KorB, it was Tom’s idea and initiative to investigate KorA as well. That decision proved to be key to our discovery.
Biology is like a treasure chest – you know you’ll find something unknown inside, but you’re never quite sure what it will be until you open it. And often, what you find is more amazing than you imagined.
Q: Were you surprised to find such complexity in a plasmid, which is a relatively simple genetic structure?
A: In a way, yes. It was delightful to uncover such intricate regulatory mechanisms. But after years of working in biology, I’ve come to expect surprises. Biology is like a treasure chest – you know you’ll find something unknown inside, but you’re never quite sure what it will be until you open it. And often, what you find is more amazing than you imagined.
Q: Your research involved a large international team and various advanced techniques. Can you tell us more about the collaboration?
A: Collaboration was absolutely crucial to our success. We worked with experts from multiple institutions, including Chris Thomas at Birmingham University, Fernando Moreno-Herrero with his team members Paco Balaguer-Pérez and Clara Aicart-Ramos at the Centro Nacional de Biotecnología in Spain, Seth Darst and Liz Campbell and their team members Joshua Chandanani and Sophia Burick at Rockefeller University in the USA, and Brian Chait and Dom Olinares from Rockefeller as well. Without their contributions, we couldn’t have completed this study.
Initially, my team conducted some foundational work on KorA and KorB, but we knew that we needed specialised techniques beyond our lab’s capabilities to make our findings airtight. So, we reached out to collaborators whose expertise and resources could help us push our research further.
What I loved most about this collaboration was that it wasn’t just about confirming our initial findings – our partners made discoveries that added even more depth to our understanding of the biology. It was a true team effort, and everyone played a meaningful role.
Q: What’s next for this research? Could it help in the fight against antimicrobial resistance?
A: My primary motivation has always been discovery science, and I appreciate that organisations like the Lister Institute support fundamental research. However, this work could have practical applications as well.
The mechanism we uncovered might be widespread in other antibiotic-resistant plasmids, so understanding it better could contribute to efforts to limit the spread of antimicrobial resistance. Additionally, we know that without KorB, the plasmid – and its host bacterium – cannot survive. That makes KorB a potential Achilles’ heel. Since KorB binds to a small molecule, CTP, there’s an exciting possibility that a small molecule inhibitor or drug could be designed to disrupt it – perhaps preventing the clamp from closing, for instance. This is something worth exploring in future studies.
What I loved most about this collaboration was that it wasn’t just about confirming our initial findings – our partners made discoveries that added even more depth to our understanding of the biology. It was a true team effort, and everyone played a meaningful role.
Q: Thank you for sharing your insights, Tung. Where can people learn more about your work?
A: Thank you! Those interested can read our full paper in Nature Microbiology or explore more about our research at www.tunglelab.org
