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After the advent of antibiotics in the 1940s, scientists were certain that they were on the cusp of conquering infectious diseases, their confidence bolstered by the notion that a comprehensive knowledge of bacterial pathogens was already well documented.

Then, drug resistance struck not long after the worldwide antibiotics revolution began. It was an eye-opening lesson about bacterial complexity. Now, a new complexity lesson is unfolding on another front involving the overlooked and understudied populations of small proteins that pervade the bacterial cell. These tiny molecules are intimately involved in an array of processes that range from aiding the assembly of bacterial membranes to mediating signaling activities. They are ripe to be exploited as targets in new types of antibacterial medications, a growing number of scientists say.

A critical group of small molecules attracting a spotlight reside in Escherichia coli and are intricately connected with how the bacterial cell responds to potentially lethal events.

A team of researchers at Rutgers University in New Jersey has discovered a slew of these small proteins in E. coli, and one in particular, which the team has christened with an unusual name, YoaI. The Rutgers team found that YoaI mediates cross-talk across a dual-component signaling system as stress signaling widens amid plunging levels of magnesium, a finding that expands the known repertoire of stress-induced small proteins that regulate signaling in E. coli.

Researchers compared YoaI to other small proteins in the bacterium and constructed a detailed portrait of these molecules and their roles when levels of the mineral precipitously drop.

The research is in Science Signaling and opens a new window of understanding into proteins that have long been overshadowed by research focused on other components of the bacterial cell.

The Rutgers group of collaborators found that the E. coli genome encodes at least 150 small proteins, most of which are functionally uncharacterized. Small bacterial proteins are defined as having fewer than 50 amino acids. As tiny as they are in size, they can have a titanic impact on the life and survival of E. coli, aiding the response to stress from the environment.

"Living organisms sense and respond to environmental stressors through a wide variety of gene regulatory mechanisms," writes Dr. Sangeevan Vellappan, lead author of the investigation and a bioinformatics researcher on the Rutgers team.

In a look at past research on small proteins in bacterial cells, Vellappan noted that while "some of these mechanisms involving regulators such as transcription factors and small RNAs have been investigated in depth, other regulatory pathways essential for stress adaptation—like the proteins investigated in this study—remain less well understood."

"We identified and characterized 17 small proteins induced in E. coli during magnesium starvation," Vellappan continued, noting that starvation was induced in the experiments.

Magnesium is key to E. coli's survival. When starved of the mineral, the team was able to trace the stress signals that the bacteria propagated.

In their experiments, Vellappan and colleagues found that magnesium depletion threatened multiple critical processes, such as the synthesis of DNA, RNA and proteins. Stress responses rose in E. coli as soon as magnesium declined. According to data in the study, the team was able to pinpoint the emergence of the 17 small proteins by using a specialized type of RNA sequencing called ribosome profiling.

Small proteins, Vellappan emphasized, are emerging as key subjects of research. Scientists worldwide are seeking a deeper level of understanding about the myriad mechanisms underlying the function of E. coli (and other bacteria) with the aim of developing deeper insights—but at the same time, seeking sites of vulnerability for new drug targets.

While most strains of E. coli are harmless, pathogenic strains of the bacteria, such as Shiga toxin-producing E. coli—STEC—can cause life-threatening diarrhea. Multiple other pathogenic strains also cause life-threatening diarrhea and deadly bloodstream infections. Uropathogenic E. coli—UPEC—can lead to serious urinary tract infections.

E. coli infections are major public health concerns, and multidrug resistance has increased the bacteria's lethality. Globally, the bacteria are associated with an estimated 500,000 deaths annually, according to a 2022 study in The Lancet.

But the Rutgers team was focused on the intricacies of signaling under conditions of magnesium depletion. In that quest, they connected several of the 17 small proteins to the magnesium-sensitive two-component PhoQ-PhoP signaling system. In this part of their research, they confirmed that deleting or overexpressing small proteins disrupted the growth of E. coli and its ability to adapt to low magnesium.

The team then focused on YoaI, which increased under low magnesium but wasn't linked to PhoQ-PhoP signaling. Instead, this protein activated another two-component system, EnvZ-OmpR, that controls how E. coli responds to a different type of stress, namely osmotic fluctuation.

Overall, the research by Vellappan and colleagues expands scientific knowledge of small proteins involved in stress adaptation in E. coli and uncovers the importance of the single small protein, YoaI. Equally important, the new investigation bolsters knowledge, in general, about the role played by small bacterial proteins that coordinate stress responses in bacteria.

The team sees a more pragmatic side to their investigations—how additional research can deepen insights into E. coli's signaling cascade. "Our results suggest that these proteins play broader roles in coordinating stress responses, reflecting the interconnected nature of cellular stress networks rather than strictly compartmentalized pathways responding to specific stressors," Vellappan concluded.

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