(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Collective polarization dynamics in bacterial colonies signify the occurrence of distinct subpopulations [1] ['Marc Hennes', 'Institute For Biological Physics', 'Center For Molecular Medicine Cologne', 'University Of Cologne', 'Cologne', 'Niklas Bender', 'Tom Cronenberg', 'Anton Welker', 'Berenike Maier'] Date: 2023-01 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. Funding: This work was supported by the Center for Molecular Medicine Cologne ( www.cmmc-uni-koeln.de ) through grant B6 and the Deutsche Forschungsgemeinschaft ( www.dfg.de ) through grant MA3898 granted to BM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2023 Hennes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Bacteria actively maintain a negative membrane potential as part of the ion motive force. Functioning as a source of energy, ion motive force powers ATP synthesis, transport across the membrane, and membrane-standing molecular machines [1–3]. Recent studies investigating membrane potential of Escherichia coli and Bacillus subtilis at the single-cell level revealed that the membrane potential is highly dynamic and heterogeneous [4,5]. Transient hyperpolarization is associated with increased death rate [6], while depolarization has been shown to maintain viability under oxygen depletion [7] and antibiotic treatment. In particular, aminoglycoside antibiotics induce hyperpolarization [6,8] and hyperpolarized cells tend to grow more slowly and have a higher death rate [6]. These reports provide evidence that inhibition of hyperpolarization maintains growth and viability. While there is no evidence of collective hyperpolarization in bacterial populations, spatially propagating waves of membrane depolarization have been found in B. subtilis colonies [9]. These waves coordinate the metabolic state and growth behavior between the interior and the periphery through the release of intracellular potassium via dedicated ion channels [9,10]. To our knowledge, collective dynamics of membrane potential in colonies and biofilms has not been found in other species so far. Biofilms are an abundant form of bacterial life [11]. Within biofilms, localized gradients of nutrients, oxygen, and waste provide habitat diversity [12]. One consequence of this diversity is that bacteria partition into fast growing cells at the surface of the biofilm and slowly growing cells at the center [12,13]. Slow growth tends to increase tolerance of the bacteria against different antibiotics and other stresses [12,14]. Heterogeneity and dynamics of membrane potential potentially contribute to local habitat formation and antibiotic tolerance. Well-studied biofilm-formers like Pseudomonas aeruginosa initiate biofilm formation by surface attachment of single planktonic cells and subsequent proliferation into colonies [15,16]. The irreversible transition from the planktonic state into the biofilm state is believed to occur gradually at this stage [16]. In this study, we address these dynamics using Neisseria gonorrhoeae (gonococcus), the causative agent of the second most prominent sexually transmitted disease, gonorrhea [17]. By contrast to P. aeruginosa, N. gonorrhoeae uses type 4 pilus (T4P)-driven motility to self-aggregate into surface-attached spherical microcolonies consisting of thousands of cells within a few minutes [18–20]. T4P are extracellular polymers that continuously elongate and retract [21–24]. T4P dynamics are crucial for the structure of gonococcal colonies; a tug-of-war mechanism fluidizes the colonies, introducing local liquid-like order and causing colonies to form spheres [25–30]. Colony formation protects N. gonorrhoeae against the β-lactam ceftriaxone [31] and the degree of tolerance depends on the physical properties of the colonies [29]. Within several hours, a gradient of growth rates develops in these colonies [32], but it is unclear how and at which time scale habitat diversity emerges. Such transitions are expected to occur at some point in the maturation process of the biofilm and may be linked to cell differentiation and the emergence of sleeper cells [12,14]. The spherical geometry makes gonococcal colonies an ideal system for studying the evolution of the membrane potential during maturation of freshly assembled colonies into biofilms. In this study, we focus on the dynamics of membrane potential in gonococcal colonies during early colony development. Using single-cell analysis within spherical colonies, we investigate the polarization dynamics at different stages of colony development. In freshly assembled colonies, single-cell membrane potential is heterogeneous and spatially uncorrelated. Eventually, a shell of hyperpolarized cells occurs at the colony center and travels towards the colony periphery. This event signifies a transition to collective membrane potential dynamics and correlates with reduction of growth. Behind the hyperpolarized shell, the intracellular potassium concentration increases and cells depolarize. A reaction–diffusion model strongly suggests that the dynamical pattern of oxygen concentration shapes the polarization dynamics. Application of the protein synthesis inhibitors kanamycin or azithromycin reverses the direction of the traveling shell. Within the shell, tolerance against kanamycin increases. Taken together, we show that membrane potential dynamics signifies the occurrence of habitats with different growth rates and antibiotic tolerance. In large colonies, a shell of hyperpolarized cells travels through the colony and marks the transition from volume to surface growth As the colonies grew in size, we found that the membrane potential transitioned from the uncorrelated dynamics described above to collective dynamics. Sometime after colony assembly in flow chambers, cells in the center of the colony showed increased polarization relative to the mean of the colony (Fig 2A and S2 Movie). Henceforth, we will refer to this relative increase in polarization as (collective) hyperpolarization. Collective hyperpolarization was transient and propagated radially from the center towards the edge of the colony across all cells. Notably, the shell of hyperpolarized cells rarely reached the edge and mostly became stationary a few μm away from the interface. The angular correlation coefficient shows a clear maximum at the position of the hyperpolarized shell (Fig 2B), indicating collective changes in membrane potential. The shell has a thickness of 3 to 4 μm, corresponding to 3 to 4 cell diameters. The difference of the membrane potential between the hyperpolarized cells and the cells at the colony center is ΔV m ≈ −10 mV (Fig 2C). PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 2. Traveling shell of hyperpolarized cells in large colonies. Strain wt green strain (NG194). t trans is the time point at which the membrane potential transitions to collective behavior. (A) Typical time lapse of TMRM fluorescence in flow cell, beginning 3 h after incubation. Scale bar: 5 μm. (B) Angular correlation of the intensity fluctuations as a function of radial position within the colony . The images correspond to the fluorescence images in (A) at the time points indicated by orange frames. (C) Radial membrane potential of cells in colonies at the time point where the hyperpolarized cells reside 7 μm from the edge of the colony (d: distance from the edge of the colony). (D) Colony radius R(t) normalized to an exponential function . The time axis was shifted to set t = 0 at the time when collective hyperpolarization occurs in the TMRM image. λ 0 : initial growth rate, R 0 : initial colony radius. Deviation from 1 indicates deviation from exponential growth. Black line: mean of 10 colonies from 3 different measurement days. Gray lines: individual colonies. (E) Time point of the transition to collective behavior as a function of initial colony size in static cultures (see S1 Data for raw values). https://doi.org/10.1371/journal.pbio.3001960.g002 Previously, we showed that within colonies of approximately 2 to 3 h of age, a characteristic pattern of growth rates forms whereby the rates decrease from the edge of the colony towards its center [32]. We assessed whether growth inhibition and the onset of collective membrane dynamics are correlated. Deviation from an initially exponential growth would indicate reduced growth rates within colonies. We determined the radii R of individual colonies as a function of time. As previously shown [32], the radius increases exponentially in freshly assembled colonies for approximately 2 h, i.e., , with the initial radius R 0 and the growth rate λ 0 . To detect the deviation from exponential growth, we normalized R(t) by the radial growth function of the first 2 h, yielding . R* = 1 signifies volume growth and R* < 1 surface growth. For the individual colonies, we found that the deviation from exponential growth coincided with the onset of collective hyperpolarization (Fig 2D). Prior to the formation of the hyperpolarization shell (t−t trans < 0), colony radii grew exponentially, R* ≈ 1, with an average growth rate λ 0 ≈ 0.6 h−1 in line with previous measurements [32]. After shell formation (t−t trans > 0), the normalized radius R* quickly fell below 1. Relating the instantaneous radial position of the shell with the colony radius R(t) indicates that growth cessation is limited to regions of the colony through which the hyperpolarized shell has already passed (Materials and methods, and Fig iv in S1 Text). Previously, we showed that the growth rate is slowest at the colony center while a narrow layer of cells continues to grow unperturbed [32]. The layer of growing cells [32] and the layer of cells that has not experienced transient hyperpolarization both comprise 3 to 4 cell diameters. We conclude that collective hyperpolarization in our gonococcal colonies coincides with growth arrest and marks the transition from volume to surface growth. Within a single field of view in the microscope, the colonies did not transition simultaneously into the collective state (S3 Movie). Larger colonies transitioned earlier than smaller colonies. To characterize the relation between colony size and time point of the transition systematically, colonies were grown in static cultures without continuous flow, which accelerated the transition to collective polarization. We found that indeed, that the delay decreased with increasing initial size of the colony (Fig 2E). Furthermore, with increasing initial cell density, the delay decreased (Fig v in S1 Text), suggesting that collective hyperpolarization is caused by depletion of nutrients or oxygen. In conclusion, we discovered a transition in membrane polarization dynamics from independent to collective changes in membrane potential that depends on colony size and marks the transition from volume to peripheral growth. 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