New Technology Illustrates Bacterial "Hibernation States"

Like hibernating bears, certain bacterial cells have the ability to shift into a "low-power" metabolic state in which they can stably survive without growing. This state is often seen in the context of antibiotic-resistant infections as bacteria form a sticky mass called a biofilm; this low-power state enables bacteria in biofilm cores to resist commonly used antibiotics. Studying bacteria in this context has historically posed technical challenges for researchers.

Now, researchers have developed lab-based technology to study the metabolic state of biofilm cores represented in a miniaturized liquid suspension using a highly versatile, high-throughput system. The new device employs a 96-channel potentiostat (a device to measure voltage differences) that measures the power output of each liquid well with tiny electrodes.

The work is a collaboration between Caltech's Dianne Newman, the Gordon M. Binder/Amgen Professor of Biology and Geobiology and Merkin Institute Professor; and Akihiro Okamoto of the National Institute for Materials Science in Japan and a visiting associate in biology and biological Engineering at Caltech. The findings are described in a new paper appearing in the journal Cell on October 23. Former graduate student John Ciemniecki (PhD '24) is the study's first author; graduate students Chia-Lun Ho of Hokkaido University and Richard Horak of Caltech also made important contributions to the study.

The team used the device to examine the bacterium Pseudomonas aeruginosa (a leading hospital-acquired pathogen that causes a wide range of acute and chronic infections) in its low-power metabolic state.

The Newman laboratory has studied Pseudomonas for decades. In particular, the group has identified small molecules called phenazines that the bacteria use to fuel metabolism in the absence of oxygen. As there is often no oxygen deep within infectious biofilms, phenazines are a crucial way for Pseudomonas to survive—and thus, a potentially potent target for therapeutic drugs.

Okamoto and his laboratory have studied microbial electrochemistry for many years and initially developed the 96-channel potentiostat device to study the bacterium Shewanella oneidensis, a model organism used in energy and environmental applications. Collaborating with the Newman laboratory, Okamoto realized the potential biomedical applications of the device.

"We were able to demonstrate that the measured currrent directly reflected the rate of metabolism," Okamoto says. "Combined with Dianne's methods, we found that this rate was extremely slow and amenable to mechanistic dissection."

With the experimental abilities opened up by the 96-channel potentiostat device, Newman's team could finally tackle longstanding questions about phenazines and Pseudomonas. The team compared many different conditions and various genetic mutants of Pseudomonas to determine if the bacteria were operating at their minimum metabolic threshold and understand what enzymes were necessary to conserve energy. They found that genes encoding bioenergetic machinery merging fermentative and respiratory pathways were necessary for the bacteria to maintain stable low-power stasis; without these genes, the bacteria died more rapidly.

The new technology can be used to study many different strains under a wide range of conditions.

"To our knowledge, until now, no experimental system had permitted direct, high-throughput measurements of metabolic power output when cells are metabolically active but not growing," Newman says. "We now have an opportunity to make discoveries about how maintenance metabolism works that may be generalizable to other organisms, something that is fascinating at a very basic level but also directly relevant to identifying new drug targets to combat recalcitrant infections."

The paper is titled "Mechanistic study of a low-power bacterial maintenance state using high throughput electrochemistry." Funding was provided by the National Institutes of Health, the Japan Society for the Promotion of Science, and the Japan Science and Technology Agency.