Author: Noemí Alonso
Understanding Cyclic di-GMP signalling in Pseudomonas aeruginosa
In today’s article, we aim to take a closer look at the scientific study Genetic Dissection of Cyclic di-GMP Signalling in Pseudomonas aeruginosa via Systematic Diguanylate Cyclase Disruption.
In this study, a team of researchers led by Román A. Martino and Daniel C. Volke investigated how the bacterium Pseudomonas aeruginosa produces cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a chemical compound that plays a crucial role in the survival of these bacteria.
This molecule acts as a messenger that translates external signals received by the bacteria into specific actions. It functions like a molecular switch, triggering bacterial changes such as biofilm formation for protection and directly influencing the bacteria’s ability to become virulent.
Dismantling Pseudomonas aeruginosa
These bacteria are one of the major nightmares in hospitals, as they cause chronic infections, particularly in immunocompromised patients. They can affect the lungs, respiratory tract, urinary tract, and wounds, often leading to sepsis.
Their ability to invade and cause infections is largely due to their capacity to form biofilms. In other words, the bacteria organize themselves into structured communities that adhere to surfaces, becoming highly resistant because the immune system cannot easily penetrate them. When c-di-GMP levels rise, the bacteria cluster together and increase their virulence.
Can a Single Gene Cause So Much Trouble? These bacteria do not rely on a single gene to produce enzymes capable of synthesizing c-di-GMP. Scientists have identified as many as 32 such genes. This poses a challenge: even if one gene is eliminated, the remaining 31 can still perform the same function.
How can all 32 genes deactivated at once? CRISPR/Cas9 Technology
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a gene-editing technology. It is often referred to as “molecular scissors” because it allows DNA to be cut and modified with remarkable precision. This technology is based on a natural defence mechanism used by certain bacteria.
How does this defence work? After a virus infects a bacterium, the bacterium stores fragments of the viral genome. If the virus attacks again, the bacterium produces messenger RNA from these stored fragments. This RNA recognizes the invading virus and guides Cas proteins, which cut and destroy the viral DNA, neutralizing the threat.
Scientists realized they could harness this recognition-and-cutting mechanism for genetic engineering.
How did this research team do it?
The researchers used a CRISPR variant known as base editing, which works by finding and replacing specific genetic bases. They identified all 32 genes and changed a single letter in each one, introducing a stop codon. In other words, they inserted a signal that prematurely stop enzyme production. As a result, the proteins produced by the bacteria were incomplete and non-functional.
This led to the creation of the PA14Δ33, a bacterium incapable of forming biofilms. Its ability to produce c-di-GMP was significantly reduced, although not completely eliminated. The researchers concluded that other enzymes must still be producing small amounts of c-di-GMP through alternative pathways.
What did they achieve?
The researchers observed that the new strain was not only unable to form biofilms but also showed reduced virulence. The production of chemical “weapons” used by the bacterium was diminished. Two examples are pyocyanin, a toxic pigment, and pyoverdine, a molecule capable of “stealing” iron from host cells.
To test this, the original and modified bacteria were introduced into nematode worms. The researchers found that worms infected with the new strain showed a 98% increase in survival.
Beyond gene deactivation
The researchers also reintroduced specific genes to determine whether the bacteria could recover their original functions. When the wspR gene was restored, the bacteria regained the ability to form biofilms but not their virulence. This indicated that although the 32 genes appeared to share the same role of increasing c-di-GMP levels, not all of them contributed to virulence. In other words, each enzyme has additional, specific functions.
This research breakthrough opens the door to the development of new drugs that go beyond simply killing bacteria. Instead, future therapies could genetically disable harmful bacteria. This represents a major milestone in the fight against antimicrobial resistance.
What is the role of TOLERATE in all this?
TOLERATE searches for ancient genes preserved in Arctic permafrost that could help improve the resilience of modern crops. Its mission is to identify and extract genes that have already survived extreme conditions in the past and apply them in today agriculture. The goal is to redesign rhizosphere bacteria and transform them to support crop growth.
This research provides two key tools to achieve that objective. One of TOLERATE’s aims is to introduce ancient genes into modern microorganisms. The creation of the PA14Δ32 strain is an example of how this approach can “clean” the genome of modern bacteria, turning them into suitable hosts for genes from the past. By removing existing genes, the bacteria’s native genetics no longer interfere with the introduced functions. Additionally, the successful use of CRISPR technology demonstrates that multiple genetic changes can be made simultaneously and efficiently.
Undoubtedly, these applications are vital to produce biomolecules that help plants increase their resistance.