Last week, PNAS (Proceedings of the National Academy of Sciences) (PDF) posted a paper co-authored by the late MCB professor Howard Berg and myself, a research associate in his lab. Sadly, Howard passed away in December 2021, making this paper one of his last publications. However, research sparked by his curiosity about flagellar motors will continue for many years to come.
Bacterial cells can switch between distinct free-swimming and sessile lifestyles as they encounter and interact with solid surfaces. Past research has suggested that flagellar motors are involved in sensing the bacterium’s altered motility state and triggering a complex transcriptional program that culminates with the formation of a biofilm structure.
When Howard introduced me to the idea of this project, he told me how far our mechanistic understanding of the flagellar motor has progressed (mostly due to work done in Howard Berg’s laboratory) and how he was becoming curious about the sensing and signaling capabilities of Escherichia coli’s flagellar motor. Past research, including studies from Howard’s lab, have shown that flagellar motors respond to changes in the external environment. Howard thought that the small molecule cyclic di-GMP might be part of a sensing and signaling pathway that allows the bacterium to adjust to new environments. In our new study, we visualized the cyclic di-GMP concentration changes that occur when E. coli cells encounter a solid surface.
To understand how flagellar motors sense and signal surface attachment, we developed genetically encoded fluorescent biosensors that allowed us to track dynamic cyclic di-GMP concentration changes as free swimming bacteria interact with a glass surface. E.coli cells divide rapidly, so we needed biosensors that could become functional fast in the burgeoning bacterial cells. We were fortunate for the work from MCB professor Phillipe Cluzel’s laboratory, which identified fluorescent proteins that mature quickly. The biosensors we developed were thus based on the fastest maturing fluorescent proteins and allowed for dynamic cyclic di-GMP detection in exponentially growing E.coli cells, which make very small amounts of cyclic di-GMP.
When we imaged wild type E. coli interacting with the glass surface of the coverslip, we found that on average E. coli increases cyclic di-GMP concentration upon attachment and that this increase depends upon a functional flagellar motor. However, the kinetics and the amplitude of the response varied stochastically between individual cells. In addition, many of the single cell responses were preceded by a spike in the internal pH of the cell. These results are consistent with the hypothesis put forth by Hug et al., which states that when bacterial cells interact with a solid surface, the proton translocating flagellar motors are stalled, which results in a transient increase of the local pH. This increase in pH activates a motor-associated enzyme to catalyze cyclic di-GMP production.
Very little is known about the path that protons take through the motor, and Howard was fundamentally interested in how the proton flow connects to torque generation and rotation of the flagellum. How proton passage through the flagellar motor modifies activities of motor-associated enzymes is yet another mystery.
by Alina Vrabioiu and Diana Crow
(PDF)
