Synthetic biologists hack into bacterial sensors

Synthetic biologists at Rice University have hacked bacterial detection with a plug-and-play system that can be used to mix and match tens of thousands of sensory inputs and genetic outputs. Technology has many implications for medical diagnosis, the study of deadly pathogens, environmental monitoring, and so on.

In a nearly six-year project, Rice bioengineer, Jeff Tabor, and his colleagues conducted thousands of experiments to show that they could systematically rewire two-component systems, the genetic circuits used by them. bacteria to detect their environment and listen to their neighbors. Their work is included in a study published this week in Nature Chemical Biology.

The Tabor group rewired the outputs of known bacterial sensors and also moved sensors between distant bacteria. More importantly, they showed that they could identify the function of an unknown sensor.

"Based on genomic analyzes, we know that there are at least 25,000 two-component systems in bacteria," said Tabor, an associate professor of bioengineering at Rice's Brown School of Engineering and senior scientist of the project. "However, for about 99% of them, we have no idea what they detect or genes they activate in response."

The importance of a new tool to unlock two-component systems is underscored by the discovery in 2018 of two strains of a deadly, multi-resistant bacterium, which uses an unknown two-component system to escape. colistin, an antibiotic of last resort. But Tabor said that the potential uses of the tool extend beyond medicine.

"It is the largest biosensor treasure of nature," he said. "Based on the exquisite specificity and sensitivity of some of the two-component systems that we understand, it is widely accepted that bacterial sensors will surpass all that humans can make with the best technology of today." 39; hui ".

Tabor explained that this is because bacterial sensors have been perfected and perfected over the course of billions of years of evolution.

"Bacteria do not have anything as sophisticated as eyes, ears or nose, but they travel between very different environments – like a leaf, an intestine or the ground – and their survival depends on their ability to detect and adapt to these changes, "he said.

"Two-component systems are what they do," Tabor said. "These are the systems they use to" see "the light," feel "the chemicals around them and" hear "the latest news from the community, which come in the form of biochemical tweets broadcast by their neighbors."

Bacteria are the most abundant life form and two-component systems have been demonstrated in virtually all bacterial genomes that have been sequenced. Most species have about two dozen sensors and some have hundreds.

There are more than half a dozen major categories of two-component systems, but they all work the same way. They have a sensor kinase component (SK) that "listens" to a signal from the outside world and, in "hearing" it, initiates a process called phosphorylation. This activates the second component, a response regulator (RR) that acts on a specific gene – turn it on or off as a switch or raise or lower it as a dial.

While the genetic code of the components is easily identifiable on a genomic analysis, the double mystery makes it almost impossible for biologists to determine what a two-component system does.

"If you do not know the signal it's detecting and the gene it's acting on, it's really hard," Tabor said. "We know either the input or the output of about 1% of two-component systems, and we know both the inputs and outputs for a lower number."

Scientists know that SKs are typically transmembrane proteins, with a detection domain, a kind of biochemical antenna, that penetrate through the outer membrane of the bacteria. Each sensor domain is designed to lock on a specific signal molecule, or ligand. Each SK has its own target ligand and it is the binding with the ligand that triggers the chain reaction that activates, disables, inhibits or inhibits a gene.

Importantly, although each two-component system is optimized for a specific ligand, their SK and RR components operate in a similar fashion. With this in mind, Sebastian Schmidl, co-lead author of the study, decided in late 2013 to try to swap the DNA binding domain, the part of the response regulator that recognizes the DNA and activates the target gene of the pathway.

"If you look at the previous structural studies, the DNA binding domain often looks like a freight that has just come out of the phosphorylation domain," Tabor said. "Because of this, we thought that DNA binding domains could work as interchangeable modules or Lego blocks."

To test the idea, Schmidl, then a DFG postdoctoral fellow in the Tabor group, rewired the components of two light sensors that Tabor's team had previously developed, one responding at the red light and the other at the green. Schmidl re-wired the red light sensor input to the output of the green light sensor at 39 different locations between the phosphorylation and DNA binding domains. To see if any of the 39 splices worked, he stimulated them with a red light and searched for a response to the green light.

"Ten of them worked the first time, and there was an optimum, a precise place where the splice really seemed to work," said Tabor.

In fact, the test worked so well that Schmidl and himself thought they might just have been lucky and laid the groundwork for two exceptionally well-matched tracks. They therefore repeated the test, first attaching four additional DNA binding domains to the same response regulator, and then attaching five DNA binding domains to the same detection pathway. Most of these re-wirings also worked, indicating that the approach was much more modular than any previously published approaches.

Schmidl, now an assistant professor of biology at the Texas Rellis Campus A & M University System in Bryan, left Rice in 2016. Principal author: Felix Ekness, Ph.D. student of Rice's program on System, Synthetic Biology and Physics (SSPB), then undertook the project by developing dozens of new chimeras and conducting hundreds of other experiments to show that the method could be used to mix and match DNA binding domains. between different species of bacteria and between different families of two-component systems.

Tabor knew that a top-notch journal would require a demonstration of the use of the technology, and the discovery of the operation of a completely new two-component system was the ultimate test. For this, Kristina Daeffler, Postdoctoral Fellow and Ph.D. Ph.D. of the SSPB. Student Kathryn Brink has transplanted seven different unknown two-component systems of Shewanella oneidensis into E. coli. They developed a new strain of E. coli for each unknown sensor and used a DNA binding domain exchange to link all their activities to the expression of green fluorescent protein.

If they did not know the entry for any of the seven, they knew that S. oneidensis had been found in a lake in northern New York state. On this basis, they chose 117 different chemicals that S. oneidensis could benefit from detection. Since each chemical had to be tested individually with each mutant and a control group, Brink had to perform and reproduce nearly 1,000 separate experiments. This effort paid off when she discovered that one of the sensors was detecting pH changes.

A genomic research of the newly identified sensor has highlighted the importance of a tool to unlock two-component systems: the pH sensor has been detected in several bacteria, including the pathogen responsible for bubonic plague.

"This shows how unlocking the mechanism of two-component systems could help us better understand and, hopefully, better treat diseases," said Tabor.

Where does Tabor adopt technology?

He uses it to extract genomes from human intestinal bacteria in order to develop new disease sensors, including inflammatory bowel diseases and cancer, in order to develop a new generation of intelligent probiotics able to diagnose and treat these diseases.