Growing Bacteria Line Up Single File, Then Buckle in Liquid Crystals

When scientists study bacteria that are not in a petri dish or a test tube but in environments that more closely mimic their actual microbial home settings, they often find delightfully unexpected behaviors. For Sujit Datta, a professor of chemical engineering, bioengineering, and biophysics at Caltech, those oddities are an invitation to apply physics to exciting new puzzles. The results reveal previously unknown ways in which bacteria organize themselves and interact with their surroundings, and suggest new questions about how the shape of bacterial colonies affects their ability to survive, grow, and even resist treatment.

Recently, Datta and his former graduate student Sebastian Gonzalez La Corte looked at bacteria growing in liquid crystal fluids—those whose molecules are elongated and all point in the same direction. This aligned state is known to occur in some biological fluids, such as certain biofilm matrices and mucus linings of the airways and gut, yet laboratory experiments typically study bacteria in fluids whose constituents have no preferred direction. Once again, the team made some unexpected observations.

The findings are described in a paper in the journal PNAS.

Datta's group previously studied and modeled bacterial growth in polymeric fluids with randomly arranged molecules. In that setting, the team found that several commonly studied bacterial species, including Escherichia coli, Vibrio cholerae (the pathogen that causes cholera) and Pseudomonas aeruginosa (a species that often causes infections in immunocompromised or hospitalized patients) grow into long cables, several cells wide, that intertwine and form a kind of "living gel."

In contrast, the new work shows that in an aligned liquid crystal fluid, bacteria build single-cell-wide chains that grow and lengthen in relatively straight lines until they suddenly buckle in an unexpected way.

When a steel beam is compressed on either end, it eventually buckles, bending gradually in an arc-like fashion along its entire length. But when the "beam" is made of bacteria in a liquid crystal fluid, its buckling is localized—with only a small section bending very tightly. Why would these bacterial beams behave so differently?

"This is a weird mechanics problem. Mechanicians have thought about thin, slender beams buckling for decades," Datta says. "But this is an odd version of that problem because this beam is made out of cells that are self-replicating."

Datta and his colleagues collaborated with applied mathematicians at the University of Wisconsin–Madison and the University of North Carolina at Chapel Hill to build a mathematical model of bacterial growth in liquid crystals. To understand the system, Datta says to consider the metaphor of matches in a matchbox; the matches are the aligned liquid crystal molecules. Now imagine the introduction of a bacterium into the system; the bacterial cell, which is much larger than the liquid crystals, could be represented by a pencil in a very large matchbox.

"The matches don't want to be bent out of their aligned orientation, so they push back. They force the pencil to be aligned with them," Datta explains. This is what scientists refer to as "bending elasticity" in the aligned liquid crystal, and it guides much of the bacterial behavior the scientists observed in the system.

Continuing the metaphor, now imagine the pencil is growing and dividing end-on to produce more pencils, which also end up aligned and form a single chain—the most energetically favorable scenario, Datta says. At the end of the growing pencil in the box, the matches must bend slightly to accommodate the width of the pencil, and that bending costs the system energy. The bending energy penalty is steeper if multiple pencils are stacked up to form a thicker chain.

All of this became very clear in the new model, which showed that the liquid crystal molecules push on the bacteria, maintaining the observed single-cell-wide chain. And in combination with the well-established physics of liquid crystal behavior, the team was able to quantitatively predict the extent to which a bacterial chain will be aligned in a liquid crystal fluid.

The bending elasticity of liquid crystals also helps explain the unusual buckling the scientists observed. When bacteria grow in liquid crystals, the high viscosity of the liquid crystal fluid causes a buildup of drag, a compressive force along the entire length of the bacterial chain. Unlike a steel beam pressed at its two ends, the chain squeezes itself: Every cell divides, so the chain lengthens, causing the fluid drag to increase until eventually the compressive force is so strong that something has to give. But the liquid crystal molecules also resist being pushed out of alignment. In the case of normal buckling, all the liquid crystal molecules along the bacterial chain would become misaligned and bend a bit along the entire length of the chain. When the buckling is more localized, the liquid crystal molecules bend a lot but only in a small region. It turns out this latter scenario is energetically less costly than the situation where all the molecules bend.

Using confocal microscopy, the researchers observed that the chain of bacterial cells buckled multiple times creating these tight little kinks. Eventually, the buckled chains built up a tangled, serpentine network.

"We know that the spatial arrangement of bacteria influences their biological function—-how they communicate with each other, how they access nutrients, how they tolerate antibiotics. That's why being able to understand what sets the morphology of a colony is important," Datta says.

The biological implications of these observed and modeled microbial behaviors are not yet known. But now that they know about this type of growth, researchers can pose and explore a host of additional testable questions, Datta says, such as whether single-cell-chain growth might help bacteria snake through tiny crevices and colonize crowded environments, for example, host tissues or environmental soils.

The growth model also suggests a possible route toward the development of engineered living materials that incorporate bacteria for use as sensors or building materials. Growing bacterial cells inside liquid crystal environments could allow biology to do the work of organizing a material at the microscopic scale, Datta says.

More broadly, Datta says, the work provides another piece of the larger puzzle of how bacterial life is shaped by the complex environments they typically inhabit. "This is yet another environmental complexity that bacteria often have to contend with, and we just didn't know how it would alter their growth as colonies," he says. "Now we know not only how it affects that, but we can make sense of it in quantitative and predictable ways."

The paper is titled "Morphogenesis of bacterial colonies in liquid crystalline environments." The co-lead authors of the paper are Sebastian Gonzalez La Corte of Princeton University and Thomas G. J. Chandler of the University of North Carolina at Chapel Hill. Additional authors are Saverio E. Spagnolie of the University of Wisconsin–Madison and Ned S. Wingreen (BS '84) of Princeton University. The work was supported by the National Science Foundation (NSF), the Camille Dreyfus Teacher-Scholar and Pew Biomedical Scholars programs, the National Institutes of Health's National Institute of General Medical Sciences, and the Center for the Physics of Biological Function at Princeton University. It was initiated in part at the Aspen Center for Physics, which is supported by the NSF.