Understanding how cells cooperate to achieve new capabilities is important since cooperation underlies the transition from single-cell life to multicellularity. This grant supports a series of experiments by Karine Gibbs, a Professor of Biology at the University of California, Berkeley, aimed at improving our understanding of the mechanisms that facilitate collective migration (swarming behavior) of the model bacterial organism Proteus mirabilis (P. mirabilis). Understanding the swarming behavior of P. mirabilis may eventually reveal fundamental principles for forming and possibly manipulating biological collectives.Professor Gibbs' work focuses on studying the role kin-recognition plays in P. mirabilis swarming behavior. Kin-recognition is a form of intercellular communication that relies on direct cell-to-cell contact. A filament carrying a toxin at its tip extends through the membrane of a P. mirabilis bacterium and punctures a neighbor's membrane, thereby delivering the toxin to the neighbor. If this neighbor-cell is genetically the same as the 'attacking' cell (i.e. it's a relative, kin) then the neighbor cell encodes the toxin as well as the antidote and the cell lives. If, however, the neighbor is not kin, then it does not encode the toxin nor the antidote and the neighbor dies.Prior studies by Professor Gibbs suggest that kin-recognition plays a role in P. mirabilis swarming and here she will use this model organism to study how collective migration is modified by two loosely-related mechanisms: intentional mutations affecting kin-recognition, and random mutations acted on by fitness-guided selection. Gibbs will leverage her prior work that correlates growth conditions with bacterial colonies exhibiting different levels of collectivity. Specifically, by varying bacterial growth conditions Gibbs is able to create two types of bacterial communities corresponding to fast/cooperative collective migration on the one hand and slow/independent migration on the other. Professor Gibbs will use her recipes for creating fast and slow swarming communities to pursue a research project with three aims.Under Aim 1, she will develop quantitative, physical metrics that can be used to distinguish fast-swarming colonies from slow-swarming colonies. Experiments involving microscopy and subsequent image-analysis will establish quantitative descriptors of cell morphology, physiology, and motility for both single-cells and for the cell-groups observed to facilitate fast swarming. Quantitative descriptors will be developed both for single-cells and for cell-groups in both fast and slow colonies. Potential descriptors include cell area, curvature, instantaneous speed, location and trajectory, adjacency to other cells, and the number of interactions over time, among others.Under Aim 2, Gibbs will use the quantitative descriptors developed in Aim 1 to assess whether and how mutations affecting kin-recognition influence collective migration. Experiments will be performed for two 'environments': growth conditions that enable fast/cooperative swarming and growth conditions that inhibit fast/cooperative swarming (thereby favoring slow/independent migration). The experimental approach involves comparing 'regular', unmutated P. mirabilis to P. mirabilis strains with mutations that disrupt the toxin secretion system used in kin-selection. This will allow Gibbs to test the hypothesis that kin-recognition provides a fitness advantage in environments where efficient swarming is possible but not in environments where independent behaviors dominate (efficient swarming not possible).Under Aim 3, Professor Gibbs will perform experiments intended to identify genes that promote collective fitness; specifically, the ability to participate in efficient swarming. Random mutations arise at some frequency and if you start with a mutant P. mirabilis that can grow but not swarm, the bacterial population will increase (growth) but will largely be constrained to its initial location (no swarming migration). As the number of bacteria increases, eventually a mutant cell will arise that has acquired a mutation that restores the ability to swarm. This mutant will swarm away from the static population, identifying itself by spatial separation and thereby allowing experimenters to capture and genetically sequence this mutant. The researchers will then determine the genome-location of the swarm-enabling mutation as well as the alterations to the descriptors of collective vs. independent behavior.