Uncovering Hidden pH Sensing Capabilities in Microbial Cultures

In a study led by Sarah Worthan, Ph.D., a postdoctoral researcher in the Behringer Lab at Vanderbilt University, scientists successfully developed microbial cultures that have the ability to sense changes in pH, enabling rapid responses to environmental fluctuations.

In addition to highlighting the power of laboratory-driven evolution, this discovery also led to the discovery of similar mutations in nature in emerging pathogens and coral symbionts, organisms that adapt to harsh pH changes in their environment and are otherwise difficult to study.

The new paper, “Evolution of pH-sensitive transcription termination in Escherichia coli during adaptation to long-term repeated starvation,” was published in the Proceedings of the National Academy of Sciences September 19, 2024. The main result of this work is the discovery of a mutation in populations of independently evolved bacteria that occurs when bacteria are exposed to cycles of feast and famine.

According to the study, this mutation, where an arginine amino acid is replaced by a histidine, occurred on the Rho protein, which is involved in RNA transcription termination. Arginine-to-histidine mutations have also been observed in cancers and have been shown to confer an adaptive pH-sensing ability to oncogenic proteins. In bacteria, these arginine-to-histidine mutations can sense pH and alter the activity of the Rho protein to have a rapid impact on how genes are expressed.

Developed in the laboratory

Co-author Benjamin Bratton, Ph.D., assistant professor of pathology, microbiology and immunology at VU Medical Center, led the imaging for the lab experiments and analysis of the pH tests while co-author Marc Boudvillain, Ph.D., CNRS research director at the Centre de Biophysique Moléculaire in Orléans, France and a chemist by training, led biochemical experiments demonstrating altered Rho activity in pH environments.

According to Megan Behringer, Ph.D., assistant professor of biological sciences and principal investigator of the study, “This rho gene mutation has appeared repeatedly in our laboratory evolution cultures. We looked at many phenotypes and had difficulty identifying the specific effects of the Rho mutation. We then contacted Marc, who asked if we had considered that the effects might vary depending on pH.”

“That’s when we went back to our genomic data and noticed that every mutation in the rho gene was associated with a mutation in the ydcI gene. Not much is known about this gene, but very recent studies suggest that it may play a role in pH homeostasis. Marc offered to test our Rho protein in vitro for pH and when he and Mildred (co-author) came back with the results, we started to piece together the whole story.”

“Dr. Behringer contacted me a few weeks after I opened my lab at Vanderbilt with this interesting observation about solution pH, but he wondered if there was a way to measure pH inside individual cells,” Bratton observed.

“Measuring the physiology of individual bacterial cells is one of the core competencies of the Bratton lab, and this collaboration has been great. Although bacteria interact with each other through their extracellular environment, individual cells have some control over their intracellular environment.”

Boudvillain added: “We were pleased to contribute to the biochemistry experiments showing that the Arg-to-His mutation indeed regulates Rho activity in a pH-dependent manner in vitro.”

Found in nature

After the laboratory experiments, the team went looking for these mutations in natural systems.

According to Behringer, “We found it in this neglected pathogen, Bartonella baciliformis, which causes Carrion’s disease in the Andean valleys of South America. This species of bacteria was already known for its pH sense because it must quickly adapt from the high pH of the insect gut to the neutral pH of human blood when transmitted by its vector, the sandfly.”

These findings also have implications for the world of marine sponges. According to the study, ocean pH forms gradients in particular areas, such as inside hydrothermal vents or inside sponge bodies. Microorganisms that live in and around these areas must be able to adapt quickly to both environments. Their evolved gene expression allows for this transition; however, climate change could seriously alter this dynamic.

“If the ocean pH starts to become more like that of the sponge, it could put bacteria and their symbionts at risk,” Behringer said. “The bacteria could lose the environmental signal that induces the correct behavior in their current environment.”

Boudvillain said the collaboration began as a happy accident. Behringer first contacted Boudvillain to see an additional figure that had disappeared from a website. After making contact, the two realized their research trajectories matched well.

“I was very pleased to be part of this interdisciplinary work. I learned a lot from Ben, Sarah and Megan,” he said. “It was a great pleasure and opportunity to work with these dynamic young colleagues.”