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DynaTweezers: Elucidating the Molecular Mechanisms of Bacterial Gene Silencing using Tethered Particles and Optical Tweezing

Periodic Reporting for period 2 - DynaTweezers (DynaTweezers: Elucidating the Molecular Mechanisms of Bacterial Gene Silencing using Tethered Particles and Optical Tweezing)

Reporting period: 2020-06-01 to 2021-05-31

The rise of bacterial resistance to antimicrobial medication is a growing concern and each year an increasing number of hospitals report encountering bacteria resistant to all known antibiotics. Enteric (intestinal) bacteria in particular, have been identified as a reservoir for antibiotic resistant genes. An alarming example are bacteria that produce a single enzyme that can render the bacteria resistant to dozens of frontline antibiotics. The spread of antimicrobial resistance, both within bacteria species and between species, is greatly accelerated by the process of horizontal gene-transfer. Horizontal gene-transfer is a mechanism by which bacteria incorporate genetic elements obtained from other organisms into their own chromosome, and plays a major role in spreading virulence genes and antimicrobial-resistance genes.

Getting new DNA from other organisms can allow the bacteria to adapt rapidly to external conditions. This can increase their fitness, or can decrease their fitness if harmful genes are expressed. To protect themselves from foreign (Xenogeneic) DNA, bacteria have developed an immune system based on proteins that bind to the bacterial DNA, the nucleoid: nucleoid associated proteins (NAPs). NAPs find and silence the expression of genetic material acquired from other organisms. However, how this silencing works is still unclear. The aim of my work is to clarify the molecular mechanisms by which NAPs (and related co-regulatory) proteins interact with DNA to silence the expression of this DNA. Several mechanisms have been hypothesized, based on experimental observations, but it is unclear which mechanism is dominant/correct and under which conditions. Understanding the mechanism by which NAPs silence the expression of genetic material may facilitate the development of medication with the specific aim of repressing or reverting antimicrobial resistance and virulence gene expression in bacteria. My work therefore aims to figure out how this works exactly.

I develop a new biophysical method with a basis in well-established techniques to characterize these silencing mechanisms. Applying this technique, I can systematically characterize the effect that NAPs have on the transcription of specific DNA sequences, under a variety of local conditions to further our understanding of how Xenogeneic gene silencing occurs.
I have developed an improved Tethered Particle Motion (TPM) system to study the interaction between NAPs and DNA. In TPM we look at a small, micrometer sized particle bound to a surface with a piece of DNA with a microscope. The motion of the particle shows how the DNA is interacting with the NAPs. My improved TPM system is much more stable and uses a click-chemistry binding strategy to attach DNA with high efficiency to the surface. The improved system greatly increases the rate at which experimental results can be obtained, and improve the resolution with which I can observe these interactions greatly, and experiments can be performed at different temperatures. I used the improved TPM system to study the interaction between the nucleoid associated proteins H-NS and Lsr2 and DNA for different DNA sequences and at different temperatures.

In my experimental observations I find that H-NS interacts with DNA sequences in a highly structured manner in which H-NS forms a filament along the DNA, stiffening it. As a result the DNA becomes more elongated and rigid and the particle motion moves further out. The H-NS filament forms in several stages with increasing concentration of H-NS, indicating that the interaction with the DNA is structured in some way. Different percentages of DNA nucleotides CG and AT lead to a shift in the strength of the interaction, with H-NS generally binding stronger to AT-rich DNA. This general trend is not always observed so there is an effect that likely depends on the sequence of the DNA. Introducing just 6 basepairs of DNA in a sequence that H-NS is known to bind to (a nucleation site) lowers the concentration at which the the H-NS filament formation happens. Next we looked at the temperature dependence of the H-NS - DNA interaction. That interaction changes as it is used by the bacteria to sense whether it is inside or outside a host organism. We find that raising the temperature from room temperature to 38 Celsius lowers the affinity of H-NS for DNA. At 38C only a small fraction of the stiffening effect remains. The transition is gradual and at least partially reversible when the temperature is lowered, indicating that the temperature induced effect can be undone.
I have developed and demonstrated a greatly improved TPM system to study interactions between proteins and DNA. Where traditional TPM systems rely on weak antibody coupling strategy, which dissociates more rapidly at increasing temperatures and adds between 0 and 15 nm of variability to the tether length due to its orientation, the improved TPM systems creates strongly bound particles with a well-controlled tether length. The TPM system facilitates a high-density of reporter particles, the use of a wide variety of DNA sequences, high antifouling resistance, physiologically relevant elevated temperatures up to at least 45 °C, and is compatible with Mg2+ containing buffers, while samples with tethered particles can remain stable for 6 months or longer. These improvements enabled me to study sequence dependence and temperature effects on the interaction between H-NS and DNA at an unprecedented level. With this system I will continue to study the interaction between H-NS, Lsr2 and DNA in further detail. The ultimate aim is to provide enough insight in the DNA expression silencing mechanism that medicines targeting these proteins can be developed to act against the spread of antibiotic resistence.

In addition to the fundamental biophysical insight in the function of NAPs the improved TPM could form a great platform to develop sensors for small molecules (as has been demonstrated before using the traditional TPM system). These improved sensors can be applied for for example for developing medical biosensors that can be used to monitor the patient status over extended periods of time.
Overview of the system used to study the NAP - DNA interaction