Collective polarization dynamics in bacterial colonies signify the occurrence of distinct subpopulations.

Membrane potential in bacterial systems has been shown to be dynamic and tightly related to survivability at the single-cell level. However, little is known about spatiotemporal patterns of membrane potential in bacterial colonies and biofilms. Here, we discovered a transition from uncorrelated to collective dynamics within colonies formed by the human pathogen Neisseria gonorrhoeae. In freshly assembled colonies, polarization is heterogeneous with instances of transient and uncorrelated hyper- or depolarization of individual cells. As colonies reach a critical size, the polarization behavior transitions to collective dynamics: A hyperpolarized shell forms at the center, travels radially outward, and halts several micrometers from the colony periphery. Once the shell has passed, we detect an influx of potassium correlated with depolarization. Transient hyperpolarization also demarks the transition from volume to surface growth. By combining simulations and the use of an alternative electron acceptor for the respiratory chain, we provide strong evidence that local oxygen gradients shape the collective polarization dynamics. Finally, we show that within the hyperpolarized shell, tolerance against aminoglycoside antibiotics increases. These findings highlight that the polarization pattern can signify the differentiation into distinct subpopulations with different growth rates and antibiotic tolerance.

Spatiotemporal dynamics of growth and death within spherical bacterial colonies.

Bacterial growth within colonies and biofilms is heterogeneous. Local reduction of growth rates has been associated with tolerance against various antibiotics. However, spatial gradients of growth rates are poorly characterized in three-dimensional bacterial colonies. Here, we report two spatially resolved methods for measuring growth rates in bacterial colonies. As bacteria grow and divide, they generate a velocity field that is directly related to the growth rates. We derive profiles of growth rates from the velocity field and show that they are consistent with the profiles obtained by single-cell-counting. Using these methods, we reveal that even small colonies initiated with a few thousand cells of the human pathogen Neisseria gonorrhoeae develop a steep gradient of growth rates within two generations. Furthermore, we show that stringent response decelerates growth inhibition at the colony center. Based on our results, we suggest that aggregation-related growth inhibition can protect gonococci from external stresses even at early biofilm stages.

Molecular Motors Govern Liquidlike Ordering and Fusion Dynamics of Bacterial Colonies.

Bacteria can adjust the structure of colonies and biofilms to enhance their survival rate under external stress. Here, we explore the link between bacterial interaction forces and colony structure. We show that the activity of extracellular pilus motors enhances local ordering and accelerates fusion dynamics of bacterial colonies. The radial distribution function of mature colonies shows local fluidlike order. The degree and dynamics of ordering are dependent on motor activity. At a larger scale, the fusion dynamics of two colonies shows liquidlike behavior whereby motor activity strongly affects surface tension and viscosity.

Mobility of extracellular DNA within gonococcal colonies.

Transformation enables bacteria to acquire genetic information from extracellular DNA (eDNA). Close proximity between bacteria in colonies and biofilms may inhibit escape of eDNA from the colony but it also hinders its diffusion between donor and recipient. In this study, we investigate the mobility of DNA within colonies formed by Neisseria gonorrhoeae, and relate it to transformation efficiency. We characterize the penetration dynamics of fluorescent DNA into the colony at a time scale of hours and find that 300 bp fragments diffuse through the colony without hindrance. For DNA length exceeding 3 kbp, a concentration gradient between the edge and the center of the colony develops, indicating hindered diffusion. Accumulation of DNA within the colony increases with increasing DNA length. The presence of the gonococcal DNA uptake sequence (DUS), which mediates specific binding to type 4 pili (T4P) and uptake into the cell, steepens the radial concentration gradient within the colony, suggesting that the DUS reduces DNA mobility. In particular, DNA of N. gonorrhoeae containing multiple DUS is trapped at the periphery. Under conditions, where DUS containing DNA fragments readily enter the colony center, we investigate the efficiency of transformation. We show that despite rapid diffusion of DNA, the transformation is limited to the edge of young colonies. We conclude that DNA mobility depends on DNA length and specific binding mediated by the DUS, resulting in restricted mobility of gonococcal DNA. Yet gonococcal colonies accumulate DNA, and may therefore act as a reservoir for eDNA.