Bioengineers discover knob to dial up fitter cells

Cells are the fundamental units of life, but the rules that govern their successful growth and reproduction have remained mysterious.

Now, in one experiment, Stanford bioengineers show that bigger bacterial cells have an advantage over smaller ones. In a second study, they discovered that experimental microbes were able grow as fast as normal cells even with replacement parts from another species.

Both sets of experiments used E. coli, the bacterium that is publicly synonymous with food poisoning but is widely used as a model organism to study the basic processes of life. Stanford bioengineering associate professor K C Huang and colleagues described the two investigations in separate articles in Cell Reports.

In the first study, the Stanford team genetically engineered E. coli mutants to have different sizes and then tracked their success in a gladiatorial combat against smaller ancestors. They found that bigger is generally better and discovered what allows fatter cells to get the jump on their competition.

In the second study, Huang's bioengineers created E. coli mutants in which a critical part was replaced with similar parts from other species of bacteria. The goal was to discover whether the altered cells could still grow using alien parts and, if so, how the growth differed at a microscopic level.

Huang said both studies are part of a broader research program to understand the mechanisms that determine cell shape and the way that shape affects behavior.

The building blocks of cells
All cells are built from small parts called proteins, the workhorses of cellular activity. Cells use proteins to construct important structures, and E. coli cells rely primarily on one structure – the cell wall – to determine their shape and regulate many biological functions.

Proteins themselves are built in chains, with each link in the chain corresponding to a component called an amino acid. Cellular DNA serves as the blueprint to build proteins out of amino acids, link by link.

In the two studies, Stanford bioengineers made small changes to this blueprint and observed how these changes affected the growth of the entire cell. First, they changed only a single amino acid in a key E. coli protein and showed that this led to a broad range of effects, including increasing the cell's size. This was the knob.

In the second experiment, they hacked the genetic code to splice in the instructions for building a protein from a different species, like trying to run an Android app on an iPhone.

Why size matters
In the first Cell Reports paper, the Stanford team reported how changing one amino acid in a protein called MreB made E. coli fatter.

There are 347 amino acids in the chain making up MreB. By tracking how generations of E. coli evolved during many months of growth in the laboratory, the engineers zeroed in on a change to the amino acid in position 53 that made the cells more fit. To their surprise, that small change also dramatically affected cell size.

By making the tiniest of adjustments to E. coli's DNA, they instructed cells to try different amino acids at position 53 in the chain. This led to the creation of 9 different mutants across a spectrum of sizes that revealed how E. coli's size impacts its growth.

"We found a hinge that would allow us to modulate the behavior of a protein responsible for cellular structure and growth," Huang said. "We figured out how to turn it into a knob."

The next step was to test whether or not bigger cells generally gained any evolutionary advantages from their size.

In a series of experiments, the Stanford engineers had two differently sized cells compete against each other. After the cells grew and reproduced, the researchers looked to see whether there were more big cells or small cells.

They found that bigger cells outnumbered smaller competitors in all cases, and the bigger the better. The advantage was all about getting a head start on the competition: the bigger cells woke up from a dormant state more quickly when nutrients were around.

But Huang said this advantage could disappear if antibiotics were present. In that case, it would actually benefit cells to wait. Indeed, a recent study showed that ''persister'' cells survive antibiotic treatment by stopping growth and increasing the time they lie dormant.

Huang now suspects that this mechanism or one like it prevents cells from evolving to become bigger and bigger in nature. He pointed out that this work highlights the importance of studying all aspects of cell growth, not just the ability of cells to proliferate when food is abundant.

Living on borrowed parts
In the second Cell Reports paper, the engineers demonstrated the remarkable robustness of E. coli to survive and grow even when using DNA borrowed from other bacterial species.

This set of experiments focused on the protein PBP2, which is essential for constructing a functional cell wall.

To carry out this study, the bioengineers isolated the DNA that coded for related proteins in different bacterial species, including Salmonella and Vibrio cholerae.

In each case, roughly half of the amino acids were different from E. coli's native PBP2. Since changing one amino acid in MreB could drastically increase the size of E. coli, the researchers wondered if the bacterium would grow at all with an alien form of PBP2.

The Stanford team discovered that the altered E. coli still successfully built cell walls but that the size of cells could again be systematically tuned. With those changes in size came unexpected changes in structure. In particular, they found that fatter cells could have a structural twist opposite to the normal direction: making cells fatter progressively switched them from left- to right-handed, potentially affecting how they pack together as they grow in dense communities like biofilms.

This finding provided a way to separate the responsibilities of proteins such as PBP2 from proteins such as MreB, both of which play essential roles in cell wall construction. Huang notes that approaches that combine precise measurement technologies with synthetic networks of systems of proteins provide a novel strategy for dissecting the core functions of essential proteins.