Synthetic biologists at Rice University say they have developed the first method for observing the real-time activity of some of most common signal-processing circuits in bacteria, including deadly pathogens that use the circuits to increase their virulence as well as to develop antibiotic drug resistance.

Two-component systems are sensory circuits bacteria use to react to their surroundings and survive. Bacteria use the circuits, which are also known as signal transduction pathways, to sense an “unrivaled range of stimuli” from light and metal ions to pH and even messages from their friends and neighbors, said Rice bioengineering professor Jeffrey Tabor, PhD.

Tabor and postdoctoral researcher Ryan Butcher’s new optical tool for observing real-time phosphorylation reactions in two-component systems is described in a study titled, “Real-time detection of response regulator phosphorylation dynamics in live bacteria,” and published in PNAS.

“Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria,” the investigators wrote.

“Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions.

“Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.”

“Bacteria use two-component systems to activate virulence and antibiotic resistance, colonize human and plant hosts, form biofilms and foul medical devices,” said Tabor, a professor of both bioengineering and biosciences. His lab has studied two-component systems for years. In 2019, his team unveiled a biohacking toolkit that synthetic biologists could use to mix and match tens of thousands of sensory inputs and genetic outputs from the circuits.

One of the most important uses of that toolkit was unlocking the dual mystery of two-component systems. As their name implies, the circuits have two functions: sensing a stimulus outside the cell and changing the cell’s behavior in response to that stimulus.

Illustration of Rice University’s “homo-FRET” method for observing real-time phosphorylation reactions in two-component sensory systems in live bacteria. Specific stimuli outside the cell (top) initiate phosphorylation (middle), which activate response regulator proteins that form pairs (bottom right) to produce a biochemical cascade that changes the cell’s behavior. To observe phosphorylation in real-time, Rice researchers engineered strains of E. coli to produce green fluorescent tags that depolarize light from an excitation laser only when they interact as pairs (bottom right). [Figure courtesy of Ryan Butcher/Rice University]
The first component, a sensor kinase, typically protrudes through the cell’s outer wall and can only be activated by a specific chemical signal. Once triggered, it sets off a biochemical cascade, a chain reaction inside the cell that ends with the cell changing its behavior in response to the stimuli.

The first step in the cascade is phosphorylation, which results in activation of the second component of the system, the response regulator.

Though phosphorylation reactions are key in the tens of thousands of two-component systems employed in bacteria, it has been difficult to directly observe them in live bacteria. That is partly because response regulators must typically join to form pairs to carry on the biological cascade that leads to stimulus response.

“Experimental analysis of phosphorylation often requires purification of proteins from bacteria and analysis using laborious in vitro methods like gel electrophoresis,” Butcher pointed out.

Butcher created a much simpler method that relies on fluorescent protein tags and polarized fluorescent light. He engineered strains of E. coli to produce mNeonGreen fluorescent protein probes that depolarize light from an excitation laser, but only if they interact as pairs. In a number of tests, Butcher and Tabor showed their method could be used to monitor the magnitude and speed of response regulator activation under a variety of environmental conditions.

The method is called “homotypic fluorescence resonance energy transfer,” or homo-FRET for short. Tabor said researchers can use it to follow the activation of two-component systems with much higher time resolution than previously possible.

In the study, he and Butcher demonstrated the utility of homo-FRET by observing a nitrate-activated two-component system that’s known to play a role in gastrointestinal colonization by E. coli, Salmonella, and other pathogens.

“Microbiologists have known for some time that this genetic circuit is used by a number of pathogens, but we still don’t fully understand how it works,” according to Tabor.

Using their method, Tabor and Butcher discovered a previously unreported pulse of activity in the circuit in response to adding nitrate. The pulse appears to arise due to rapid activation of the two-component system followed by consumption of nitrate by the bacteria and corresponding deactivation.

“That’s a window into how this circuit works, and it’s the kind of thing that would have been much more difficult to pin down using previous methods,” Tabor explained. “With homo-FRET we can watch the circuit respond to changing nitrate levels as it’s happening.

“We think homo-FRET can be used to engineer biosensors that respond 10 times faster than current alternatives, and that we and others will be able to use it to make new discoveries in a range of other bacterial pathways.”