Quotation: Hennes M, Bender N, Cronenberg T, Welker A, Maier B (2023) Collective polarization dynamics in bacterial colonies signify the incidence of distinct subpopulations. PLoS Biol 21(1):
e3001960.
https://doi.org/10.1371/journal.pbio.3001960
Educational Editor: Victor Sourjik, Max Planck Institute for Terrestrial Microbiology: Max-Planck-Institut fur terrestrische Mikrobiologie, GERMANY
Obtained: August 23, 2022; Accepted: December 15, 2022; Revealed: January 18, 2023
Copyright: © 2023 Hennes et al. That is an open entry article distributed beneath the phrases of the Artistic Commons Attribution License, which allows unrestricted use, distribution, and replica in any medium, offered the unique writer and supply are credited.
Information Availability: All related knowledge are inside the paper and its Supporting Info recordsdata.
Funding: This work was supported by the Heart for Molecular Drugs Cologne (www.cmmc-uni-koeln.de) by grant B6 and the Deutsche Forschungsgemeinschaft (www.dfg.de) by grant MA3898 granted to BM. The funders had no position in examine design, knowledge assortment and evaluation, choice to publish, or preparation of the manuscript.
Competing pursuits: The authors have declared that no competing pursuits exist.
Abbreviations:
LHS,
left-hand aspect; PCA,
protocatechuic acid; PCD,
protocatechuate-dioxygenase; ThT,
thioflavin; TMRM,
tetramethylrhodamine methyl ester; T4P,
kind 4 pilus
Introduction
Micro organism actively preserve a detrimental membrane potential as a part of the ion driving force. Functioning as a supply of vitality, ion driving force powers ATP synthesis, transport throughout the membrane, and membrane-standing molecular machines [1–3]. Latest research investigating membrane potential of Escherichia coli and Bacillus subtilis on the single-cell degree revealed that the membrane potential is very dynamic and heterogeneous [4,5]. Transient hyperpolarization is related to elevated loss of life price [6], whereas depolarization has been proven to take care of viability beneath oxygen depletion [7] and antibiotic remedy. Specifically, aminoglycoside antibiotics induce hyperpolarization [6,8] and hyperpolarized cells are likely to develop extra slowly and have the next loss of life price [6]. These stories present proof that inhibition of hyperpolarization maintains progress and viability. Whereas there isn’t any proof of collective hyperpolarization in bacterial populations, spatially propagating waves of membrane depolarization have been present in B. subtilis colonies [9]. These waves coordinate the metabolic state and progress habits between the inside and the periphery by the discharge of intracellular potassium through devoted ion channels [9,10]. To our information, collective dynamics of membrane potential in colonies and biofilms has not been present in different species to this point.
Biofilms are an ample type of bacterial life [11]. Inside biofilms, localized gradients of vitamins, oxygen, and waste present habitat variety [12]. One consequence of this variety is that micro organism partition into quick rising cells on the floor of the biofilm and slowly rising cells on the heart [12,13]. Gradual progress tends to extend tolerance of the micro organism in opposition to completely different antibiotics and different stresses [12,14]. Heterogeneity and dynamics of membrane potential doubtlessly contribute to native habitat formation and antibiotic tolerance. Nicely-studied biofilm-formers like Pseudomonas aeruginosa provoke biofilm formation by floor 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 happen step by step at this stage [16]. On this examine, we tackle these dynamics utilizing Neisseria gonorrhoeae (gonococcus), the causative agent of the second most outstanding sexually transmitted illness, gonorrhea [17]. Against this to P. aeruginosa, N. gonorrhoeae makes use of kind 4 pilus (T4P)-driven motility to self-aggregate into surface-attached spherical microcolonies consisting of hundreds of cells inside a couple of minutes [18–20]. T4P are extracellular polymers that repeatedly elongate and retract [21–24]. T4P dynamics are essential for the construction of gonococcal colonies; a tug-of-war mechanism fluidizes the colonies, introducing native liquid-like order and inflicting colonies to kind spheres [25–30]. Colony formation protects N. gonorrhoeae in opposition to the β-lactam ceftriaxone [31] and the diploma of tolerance relies on the bodily properties of the colonies [29]. Inside a number of hours, a gradient of progress charges develops in these colonies [32], however it’s unclear how and at which period scale habitat variety emerges. Such transitions are anticipated to happen sooner or later within the maturation technique of the biofilm and could also be linked to cell differentiation and the emergence of sleeper cells [12,14]. The spherical geometry makes gonococcal colonies a super system for finding out the evolution of the membrane potential throughout maturation of freshly assembled colonies into biofilms.
On this examine, we deal with the dynamics of membrane potential in gonococcal colonies throughout early colony growth. Utilizing single-cell evaluation inside spherical colonies, we examine the polarization dynamics at completely different phases of colony growth. In freshly assembled colonies, single-cell membrane potential is heterogeneous and spatially uncorrelated. Ultimately, a shell of hyperpolarized cells happens on the colony heart and travels in direction of the colony periphery. This occasion signifies a transition to collective membrane potential dynamics and correlates with discount of progress. Behind the hyperpolarized shell, the intracellular potassium focus will increase and cells depolarize. A response–diffusion mannequin strongly means that the dynamical sample of oxygen focus shapes the polarization dynamics. Software of the protein synthesis inhibitors kanamycin or azithromycin reverses the course of the touring shell. Throughout the shell, tolerance in opposition to kanamycin will increase. Taken collectively, we present that membrane potential dynamics signifies the incidence of habitats with completely different progress charges and antibiotic tolerance.
In freshly assembled colonies, the membrane potential of neighboring cells is uncorrelated
Pushed by kind 4 pili, N. gonorrhoeae cells actively assemble into colonies comprising hundreds of micro organism [18,29]. We characterised the membrane potential of single cells inside freshly assembled colonies rising in stream chambers that repeatedly present recent medium. The fluorescence depth of the Nernstian dye TMRM was decided utilizing confocal microscopy. Relying on {the electrical} potential Vm throughout the internal membrane, the cationic dye partitions between the cytoplasm and the extracellular area [4]. The fluorescence intensities inside and outdoors of the cells had been used to measure the membrane potential as described within the Supplies and strategies. For the reason that membrane potential is detrimental, a rise in fluorescence depth signifies a rise in polarization, i.e., the potential turns into extra detrimental.
We discovered that the TMRM sign in younger colonies is very heterogeneous (Fig 1A and Fig i in S1 Textual content) From the distinction in TMRM fluorescence intensities outdoors of and inside the cells, we derived the membrane potential Vm of single cells. Potential pitfalls of this quantification are mentioned within the Supplies and strategies. Vm reveals a Gaussian distribution (Fig 1B) with a imply worth of Vm = −95 mV which is barely decrease than the worth obtained for single cells [33]. With time, the imply membrane potential of cells within the colony decreases by roughly 15 mV, indicating regular depolarization of the inhabitants (Fig 1B). The distribution of Vm departs from the preliminary Gaussian form in direction of a distribution with heavier tails at bigger potentials, attributable to a randomly distributed subpopulation sustaining robust polarization (Fig 1A and 1B).
Fig 1. Heterogeneity of membrane potential in small colonies shaped at completely different time factors.
Pressure wt inexperienced (NG194), stream chamber. (A) Typical fluorescence intensities from confocal slices by colony heart. Scale bar: 5 μm. (B) Likelihood density perform (pdf) of membrane potential of single cells at Δt = 0, 90 min, 180 min. (>4,000 cells for every time level). (C) Angular correlation of the depth fluctuations inside the colony proven in (A) (shade coded) as a perform of the angular place distinction Δφ and the gap d from the sting of the colony (see S1 Information for uncooked values).
On the single-cell degree, transient modifications in polarization have been reported [4–6,8,34,35]. To seek out out whether or not the membrane potential is dynamic on the degree of particular person cells inside colonies, we tracked the TMRM sign of single cells. To make sure that fluctuations in TMRM depth should not brought on by vertical motion of micro organism, we concurrently tracked the sfGFP sign of the respective cells (pressure wt inexperienced, Desk i in S1 Textual content). A small fraction of cells experiences occasions of transient depolarization (Figs iiA and iiB in S1 Textual content) or hyperpolarization (Figs iiC and iiD in S1 Textual content and S1 Film) that final for about 1 to 2 min. For all noticed occasions, we discover that transient modifications in polarization (roughly 15 mV change) are uncorrelated between neighboring cells. To quantify correlations in modifications of the membrane potential, we outlined a measure of how strongly deviations from the imply TMRM fluorescence depth, I(t,x,y)−〈I〉, are spatially correlated between cells. 〈I〉 is the imply depth of the colony at time t. Specifically, we calculated the 2D polar correlation perform at completely different depths d of the colony (d = 0 μm on the edge). This perform measures the correlation power of the TMRM fluorescence sign in angular course. We analysed Icorr for 20 colonies and located little correlation (Fig 1C, additional instance in Fig iii in S1 Textual content). We be aware that gonococci are likely to kind diplococci whose membrane potential seems correlated over a time scale 1 h. This evaluation confirms that the dynamics of transient modifications in mobile membrane potential happens independently between neighboring cells.
We conclude that in freshly assembled colonies, the membrane potential of single cells is heterogeneous and spatially uncorrelated. Particular person cells transiently hyper- or depolarize on the time scale of minutes whereas many of the inhabitants step by step depolarizes at a time scale of hours.
In massive colonies, a shell of hyperpolarized cells travels by the colony and marks the transition from quantity to floor progress
Because the colonies grew in dimension, we discovered that the membrane potential transitioned from the uncorrelated dynamics described above to collective dynamics. Someday after colony meeting in stream chambers, cells within the heart of the colony confirmed elevated polarization relative to the imply of the colony (Fig 2A and S2 Film). Henceforth, we are going to consult with this relative improve in polarization as (collective) hyperpolarization. Collective hyperpolarization was transient and propagated radially from the middle in direction of the sting of the colony throughout all cells. Notably, the shell of hyperpolarized cells not often reached the sting and largely turned stationary just a few μm away from the interface. The angular correlation coefficient reveals a transparent most on the place of the hyperpolarized shell (Fig 2B), indicating collective modifications in membrane potential. The shell has a thickness of three to 4 μm, corresponding to three to 4 cell diameters. The distinction of the membrane potential between the hyperpolarized cells and the cells on the colony heart is ΔVm ≈ −10 mV (Fig 2C).
Fig 2. Touring shell of hyperpolarized cells in massive colonies.
Pressure wt inexperienced pressure (NG194). ttrans is the time level at which the membrane potential transitions to collective habits. (A) Typical time lapse of TMRM fluorescence in stream cell, starting 3 h after incubation. Scale bar: 5 μm. (B) Angular correlation of the depth fluctuations as a perform of radial place inside the colony . The photographs correspond to the fluorescence photos in (A) on the time factors indicated by orange frames. (C) Radial membrane potential of cells in colonies on the time level the place the hyperpolarized cells reside 7 μm from the sting of the colony (d: distance from the sting of the colony). (D) Colony radius R(t) normalized to an exponential perform
. The time axis was shifted to set t = 0 on the time when collective hyperpolarization happens within the TMRM picture. λ0: preliminary progress price, R0: preliminary colony radius. Deviation from 1 signifies deviation from exponential progress. Black line: imply of 10 colonies from 3 completely different measurement days. Grey strains: particular person colonies. (E) Time level of the transition to collective habits as a perform of preliminary colony dimension in static cultures (see S1 Information for uncooked values).
Beforehand, we confirmed that inside colonies of roughly 2 to three h of age, a attribute sample of progress charges kinds whereby the charges lower from the sting of the colony in direction of its heart [32]. We assessed whether or not progress inhibition and the onset of collective membrane dynamics are correlated. Deviation from an initially exponential progress would point out decreased progress charges inside colonies. We decided the radii R of particular person colonies as a perform of time. As beforehand proven [32], the radius will increase exponentially in freshly assembled colonies for about 2 h, i.e., , with the preliminary radius R0 and the expansion price λ0. To detect the deviation from exponential progress, we normalized R(t) by the radial progress perform of the primary 2 h, yielding
. R* = 1 signifies quantity progress and R* < 1 floor progress. For the person colonies, we discovered that the deviation from exponential progress coincided with the onset of collective hyperpolarization (Fig 2D). Previous to the formation of the hyperpolarization shell (t−ttrans < 0), colony radii grew exponentially, R* ≈ 1, with a mean progress price λ0 ≈ 0.6 h−1 consistent with earlier measurements [32]. After shell formation (t−ttrans > 0), the normalized radius R* shortly fell beneath 1. Relating the instantaneous radial place of the shell with the colony radius R(t) signifies that progress cessation is proscribed to areas of the colony by which the hyperpolarized shell has already handed (Supplies and strategies, and Fig iv in S1 Textual content). Beforehand, we confirmed that the expansion price is slowest on the colony heart whereas a slender layer of cells continues to develop unperturbed [32]. The layer of rising cells [32] and the layer of cells that has not skilled transient hyperpolarization each comprise 3 to 4 cell diameters. We conclude that collective hyperpolarization in our gonococcal colonies coincides with progress arrest and marks the transition from quantity to floor progress.
Inside a single area of view within the microscope, the colonies didn’t transition concurrently into the collective state (S3 Film). Bigger colonies transitioned sooner than smaller colonies. To characterize the relation between colony dimension and time level of the transition systematically, colonies had been grown in static cultures with out steady stream, which accelerated the transition to collective polarization. We discovered that certainly, that the delay decreased with growing preliminary dimension of the colony (Fig 2E). Moreover, with growing preliminary cell density, the delay decreased (Fig v in S1 Textual content), suggesting that collective hyperpolarization is brought on by depletion of vitamins or oxygen.
In conclusion, we found a transition in membrane polarization dynamics from unbiased to collective modifications in membrane potential that relies on colony dimension and marks the transition from quantity to peripheral progress.
Within the wake of the hyperpolarized shell, intracellular Ok+ ranges improve and cells depolarize
After collective hyperpolarization has handed, cells depolarize barely however considerably (Figs 2C and 3A) and transient modifications in polarization as proven in Fig ii in S1 Textual content disappear. In earlier stories, potassium flux has been proven to be tightly correlated with modifications in membrane potential [7,9]. We addressed the query whether or not Ok+ ions are concerned within the membrane potential dynamics of gonococcal colonies. To this finish, we utilized the Ok+– selective ionophore valinomycin to static cultures as soon as the hyperpolarized shell had shaped. Addition of valinomycin eliminated hyperpolarization and the TMRM fluorescence was homogeneously decreased all through the colony (Fig iv in S1 Textual content). These outcomes point out that inhibition of the Ok+ gradient abolishes the attribute membrane potential sample.
Fig 3. Membrane depolarization correlates with the inflow of Ok+ ions.
Pressure wt* (NG150), static tradition. (A) Time lapse of TMRM fluorescence and (B) the Ok+ reporter IPG-2 AM throughout the equator of a colony. Scale bar: 5 μm. (C) Depth profiles alongside the ROI proven in (A) and (B) (see S1 Information for uncooked values).
To research the dynamics of intracellular Ok+, we stained wt* colonies with TMRM and a reporter for intracellular Ok+ focus, IPG-2 AM (Fig 3A–3C). We discovered that the IPG-2 AM fluorescence depth strongly elevated within the central area after the shell of hyperpolarized cells had handed, demonstrating that the focus of potassium ions elevated. This discovering means that depolarization is no less than partially brought on by an inflow of Ok+ ions. We can’t exclude, nonetheless, that different ionic species are concerned within the formation of the spatial polarization sample.
In abstract, after the hyperpolarization shell has handed, cells in its wake expertise an inflow of potassium ions and the membrane potential weakens.
A response–diffusion mannequin of oxygen uptake captures the dynamics of shell propagation
Subsequent, we investigated the underlying mechanisms of shell formation and propagation. The onset of collective hyperpolarization relies on colony dimension (Fig 2E), strongly suggesting that it’s associated to a neighborhood gradient increase inside the colony. The truth that the time level of collective polarization additionally relies on the entire focus of cells (Fig v in S1 Textual content) signifies that depletion of a progress useful resource triggers collective hyperpolarization. The diploma of oxygenation is regarded as an essential issue that influences the chemistry and physiology of a microenvironment [13,14]. As a result of biofilm morphology shapes the oxygen focus profile [36], we count on that oxygen deprivation is strongest within the heart of the colony, the place the hyperpolarization shell originates. Hypoxia has been proven to elucidate heterogeneous progress in biofilms utilizing response–diffusion idea [37].
To evaluate whether or not oxygen gradient formation can clarify the dynamics of the hyperpolarized shell, we first examined whether or not a 2D response–diffusion mannequin (see Supplies and strategies) predicts the options of shell propagation on the noticed time scales. In our mannequin, the oxygen gradient modifications with time as a result of cells proliferate inflicting colony progress (Fig 4A). We assume that the hyperpolarized shell is triggered when the oxygen focus c(t,x,y) falls beneath a vital worth c*. Due to this fact, in our simulations, we tracked iso-concentration strains akin to the focus c*, which we assume to suggest the place of the hyperpolarized shell propagation. To account for progress arrest, we set the proliferation price of cells behind these circles to zero. Moreover, we assume that the oxygen uptake price is decreased on this space by an element ε (between 0 and 1).
Fig 4. Simulation of oxygen gradients and addition of other electron acceptors counsel that oxygen depletion triggers collective hyperpolarization.
(A) Time lapse of simulated oxygen focus area. Shade code: oxygen focus . Black circles: colony radius R(t). Pink circles: radial distance riso of the iso-concentration line at focus c*. Scale bar: 20 μm. (B) Simulated propagation of the riso circle akin to c* = 0.8c0 (curves from simulations are proven for the uptake parameters ε = 0.1, 0.5, and 0.9). (C) Experimental propagation of transient hyperpolarization shells. (Means and customary errors over 20 colonies.) (D) Time lapse of TMRM sign for a colony (pressure wt*, Ng150) in static tradition supplemented with 5 mM of NaNO2. Scale bar: 10 μm. (E) Time lapse of TMRM sign for a colony (pressure wt*) in static tradition with out complement. Scale bar: 10 μm. (F) Time lapse of TMRM sign for a colony (pressure wt*) in static tradition supplemented with the oxygen scavenging system PCA and PCD (see S1 Information for uncooked values). Scale bar: 5 μm. PCA, protocatechuic acid; PCD, protocatechuate-dioxygenase.
We discovered that the propagation of iso-concentration circles qualitatively predicts the experimental dynamics of the hyperpolarization shell. Circles of focus c* begin on the heart of the colony and propagate outward at near fixed velocity (Fig 4A and 4B). Because the circle approaches the sting of the colony, propagation captured by the relative place riso/R slows down and stops a number of micrometers away from the boundary. The precise distance to the interface is ruled by the free parameter ε; if cells on the colony heart devour much less oxygen, the circle propagates extra slowly and equilibrium between oxygen diffusion and uptake is reached additional contained in the colony (Fig 4B). To match the dynamics predicted by the mannequin with the experiment, we characterised the propagation dynamics of the hyperpolarized shell by figuring out the normalized radius of the shell, rhyper/R, as a perform of time (Fig 4C). In settlement with the simulations, the shell propagated with a continuing radial velocity early after the onset of hyperpolarization and subsequently slowed down because it reached the colony periphery. The early propagation velocity of 0.11 ± 0.01 μm/min and the attribute propagation time Ï„hyper = 38 ± 1 min obtained by an exponential match to the info in Fig 4C, rhyper/R = A(1−e−t/Ï„), agree with the vary of values obtained for the simulations (Fig 4B). We be aware, nonetheless, that not all parameters required for the mannequin have been characterised experimentally for N. gonorrhoeae (see Supplies and strategies), and, due to this fact, we don’t count on actual quantitative settlement between simulation and experiment.
If a focus area of a progress useful resource is certainly concerned within the onset and dynamics of the hyperpolarization shell, we count on the transiently hyperpolarized area to be broader and fewer outlined for colonies with decreased cell quantity density and/or native ordering and non-spherical geometries. On this case, common radial gradients can be smeared out. Spherical colony form and native order require the T4P retraction motor PilT [23,25,26,38] and, consequently, ΔpilT strains kind non-spherical colonies. We repeated the stream chamber experiments with ΔpilT colonies and located that polarization dynamics of early colonies was corresponding to wt* colonies. With time, the facilities of colonies depolarized following the geometry of the combination, and, as predicted, there was solely a weak and broad signature of collective hyperpolarization (Figs viiA and viiB in S1 Textual content). To seek out out whether or not this distinction between wt* and mutant colonies was brought on by lack of T4P retraction or change in colony structure, experiments with the pressure pilTWB2 that has strongly decreased T4P retraction exercise [25], however nonetheless kinds spherical colonies with native liquid-like order [25] had been carried out. pilTWB2 colonies qualitatively behaved like wt* colonies (Figs viiC and viiD in S1 Textual content), indicating that colony morphology reasonably than T4P retraction was essential for spatially correlated hyperpolarization.
Our mannequin implies that the hyperpolarization shell propagates by transient hyperpolarization and never by stream of completely hyperpolarized cells. Within the following, we argue that the noticed dynamics is qualitatively and quantitatively completely different from the radial mass stream brought on by mobile proliferation. The expansion-related velocity is minimal on the heart of the colony and reaches its most on the fringe of the colony [32] in distinction to the end result proven in Fig 4C. The expansion-related stream velocity is 4- to 5-fold decrease in comparison with the propagation velocity of the hyperpolarized shell. On the similar time, transient collective hyperpolarization is quicker than the reshuffling of caged cells induced by pili retractions [27]. Due to this fact, we exclude cell stream of completely hyperpolarized cells (both progress or pili-driven) as the reason for shell propagation.
The propagation dynamics of the hyperpolarized shell agrees effectively with the dynamics of iso-concentration rings of oxygen. At this level, nonetheless, we can’t exclude that different progress sources like glucose underlie the noticed polarization dynamics. Oxygen acts as the ultimate electron acceptor within the respiratory chain of the micro organism [39]. If oxygen triggers collective hyperpolarization, then addition of another electron acceptor ought to retard or inhibit formation of the hyperpolarized shell. We investigated this level by supplementing the medium with sodium nitrite, which acts as an electron acceptor within the truncated denitrification pathway of N. gonorrhoeae [39–41]. We discovered that colonies supplemented with sodium nitrite exhibit no transition to collective membrane potential dynamics over the time course of 4 h (Fig 4D). Conversely, management experiments persistently present collective polarization dynamics after 30 to 60 min (Fig 4E). Moreover, we uncovered colonies to the oxygen scavenger 3,4-protocatechuic acid (PCA) along with its enzyme protocatechuate-dioxygenase (PCD) [33,42] and observe a polarization sample harking back to the late phases of shell propagation (Fig 4F).
In abstract, we current robust proof that the emergence of transient hyperpolarization and collective membrane potential dynamics is triggered by oxygen depletion alongside focus gradients that kind and journey outward in direction of the sting of the colony in radial course.
Tolerance to kanamycin however to not ceftriaxone is elevated inside the hyperpolarized shell
We investigated whether or not transitioning into the collective state impacts the survivability of cells in several circumstances. Specifically, we addressed the emergence of antibiotic tolerance. On the single-cell degree, it was proven that transient hyperpolarization correlates with elevated loss of life price [6,8], suggesting that survivability decreases inside the area enclosed by the shell of hyperpolarized cells. The state of polarization influences the bactericidal impact of aminoglycosides [8,43] and depolarization inside the shell would defend the cells. Moreover, decreased cell progress impacts the killing effectivity of sure antibiotics together with β-lactams [44], predicting that cells inside the hyperpolarized shell are extra tolerant to this class of antibiotics than the cells outdoors.
First, we studied the time evolution of the fraction of lifeless cells inside untreated colonies utilizing the membrane-impermeable DNA stain SytoX. As soon as the shell of hyperpolarized cells had handed, the fraction of lifeless cells f elevated (Fig 5A). Specifically, f contained in the hyperpolarized shell was 3.5-fold larger than outdoors of the shell (Fig 5B). This discovering is in keeping with earlier research displaying that the loss of life price of particular person cells that underwent transient hyperpolarization was larger than the loss of life price of cells with out hyperpolarization [6,8].
Fig 5. Dynamics of membrane potential and fraction of lifeless cells F in colonies after 30 min of antibiotic remedy.
Pressure wt* (NG150), stream chamber. (A, C, E) Time lapse over 4 h of TMRM and SytoX fluorescence after respectively: no remedy, kanamycin remedy (MICx10, 200 μg/ml), and ceftriaxone remedy (MICx10, 0.04 μg/ml). Scale bar: 5 μm. (B, D, F) Fraction of lifeless cells f within the colonies 3–4 h later for each the area unaffected by the hyperpolarization shell (out) and the area affected (in) after respectively no remedy, kanamycin remedy, and ceftriaxone remedy. Within the handled circumstances, f* = f−<fmanagement>. A complete of 60 colonies evaluated for every situation. Backside and prime edges of the field point out the twenty fifth and seventy fifth percentiles, respectively. Whiskers prolong to essentially the most excessive knowledge factors not thought of outliers (marked as particular person factors). Central mark signifies the median of the distribution (see S1 Information for uncooked values).
Subsequent, we addressed the questions how remedy with the aminoglycoside kanamycin impacts membrane potential dynamics and the way the pre-formed polarization sample impacts kanamycin tolerance. Two-hour-old colonies had been handled with kanamycin at 10-fold MIC for 30 min. After elimination of the antibiotics, we characterised the dynamics of the membrane potential and the fraction of lifeless cells. Transient remedy with kanamycin brought about a gradual improve in polarization all through the colony, usually beginning on the fringe of the colony and transferring inward (Fig 5C). Surprisingly, for colonies which had already shaped a shell of hyperpolarized cells, kanamycin inhibited its radial development and infrequently the shell slowly traveled again in direction of the middle of the colony, the place it disappeared if it reached the middle. Related dynamics had been noticed beneath azithromycin remedy (Figs viiiA and viiiB in S1 Textual content). To seek out out whether or not tolerance elevated within the wake of the hyperpolarized shell, we in contrast the areas of the colonies that resided contained in the hyperpolarized shell throughout antibiotic remedy with the surface area. The fractions of lifeless cells had been decided 3 to 4 h after remedy, as a result of cell loss of life is detectable with SytoX at a delay of a number of hours [29]. To account for the fractions of lifeless cells within the absence of antibiotics, we subtracted the respective values (Fig 5B), . We discovered that
inside the shell was 4-fold decrease than outdoors (Fig 5C and 5D).
Lastly, we handled the colonies with the β-lactam antibiotic ceftriaxone utilizing the identical protocol described for kanamycin. In distinction to the protein synthesis inhibitors described above, ceftriaxone didn’t have an effect on the membrane potential (Fig 5E). After subtraction of the fraction of lifeless cells with out remedy, was homogeneous all through the colony (Fig 5E and 5F), displaying that the sample of membrane potential and the related progress price discount on the heart didn’t have an effect on tolerance in opposition to ceftriaxone.
In abstract, kanamycin remedy impacts the polarization sample of the colony, whereas ceftriaxone has no impact. Throughout the hyperpolarized sphere, cells had been extra tolerant in opposition to kanamycin however not in opposition to ceftriaxone.
Dialogue
Transient hyperpolarization has been reported on the single-cell degree for E. coli and B. subtilis [4,6,8]. Importantly, hyperpolarization was uncorrelated between neighboring cells in these research. Equally, the membrane potential dynamics of freshly assembled gonococcal colonies studied right here reveals no nearest neighbor correlation. This means that gonococci, whereas being a part of multicellular colonies, nonetheless behave like planktonic cells within the first few hours after meeting. Against this to earlier stories, we reveal a transition from unbiased to correlated hyperpolarization occurring as soon as the colony is massive sufficient.
Our outcomes are most in keeping with the next state of affairs. Ruled by T4P motility and T4P-mediated cell-to-cell adhesion, gonococci assemble into colonies by T4P exercise inside minutes [23]. In the course of the early stage of this colony, gonococcal habits stays uncorrelated and colony formation is totally reversible [19]. Throughout the colony, a gradient of oxygen builds up. When a vital focus of oxygen is reached, cells hyperpolarize. As soon as the oxygen focus falls beneath this vital focus, cells depolarize, are growth-inhibited, and devour much less oxygen. For the reason that colony continues to develop on the floor, the vital iso-concentration shell (signified experimentally as a hyperpolarized shell) propagates in direction of the periphery of the colony. Relying on the oxygen consumption charges and the exogenous oxygen provide focus, the shell of hyperpolarized cells turns into stationary at a selected distance away from the sting of the colony. An analytical resolution of our response–diffusion mannequin (see Supplies and strategies) predicts that this distance is said to the vital O2 focus c*, the focus on the floor of the colony, c0, and the focus cc on the heart of the colony, . Within the stream chamber, we discover rhyper/R ≈ 0.7, which entails that the vital focus is within the vary 0.5c0 < c* < c0. Below static progress circumstances, we noticed that the hyperpolarized shell is nearer to the sting of the colony (see, e.g., Fig 4E), in settlement with the O2 focus on the floor being decrease than beneath steady stream. It’s also fascinating to notice that remedy with the protein synthesis inhibitors kanamycin and azithromycin halts or reverses the propagation of the hyperpolarized shell. Because the antibiotics inhibit the expansion of the colony and doubtlessly additional cut back the consumption price of oxygen, our mannequin predicts this remark. We be aware that our investigation is restricted to early phases of biofilm formation. In theoretical research and experiments with different species, it has been proven that response–diffusion processes have an effect on the structure of biofilms [45,46]. In future research, it will likely be fascinating to search out out whether or not response–diffusion fashions additionally predict the structure of mature gonococcal biofilms. It’s tempting to invest that the impact of oxygen is extra advanced on this species as robust depletion induces colony disassembly [19].
Whereas, we will clearly exhibit that collective and transient hyperpolarization signifies the transition of quantity to floor progress and modifications within the state of antibiotic tolerance, the molecular mechanism of transient hyperpolarization stays unclear. In line with our mannequin, the O2 focus vary that causes hyperpolarization is slender. Hyperpolarization correlated with progress arrest or elevated mortality has been reported in numerous research [6–8]. One examine identifies a trigger for hyperpolarization; beneath aminoglycoside remedy, the inhibition of ribosomes frees ATP that causes the ATP synthase to work in ATP hydrolysis course, growing the proton gradient [8]. In our system, we reasonably count on the ATP ranges to be decreased as a consequence of decreased proton gradient beneath low oxygen circumstances. It is going to be very fascinating to analyze the mechanism of transient hyperpolarization in future work.
We anticipated that decreased polarization and progress price made gonococci extra tolerant in opposition to aminoglycosides and β-lactams, respectively. Absolutely the lower in polarization between the surface and the within of the hyperpolarized shell quantities to a couple millivolts solely. But, we clearly noticed that the fraction of lifeless cells is decrease inside than outdoors of the shell after transient kanamycin remedy, indicating that cells are instantly protected in opposition to this aminoglycoside. Latest work reveals that killing exercise of aminoglycosides arises from dysregulated membrane potential [8]. They discovered that in depolarized cells, aminoglycosides exert a bacteriostatic impact. For our work, this strongly means that depolarization inside the hyperpolarized shell causes tolerance in opposition to kanamycin. But, we can’t exclude that progress arrest additionally impacts tolerance. Against this, progress arrest inside the hyperpolarized shell had no affect on tolerance in opposition to the β-lactam ceftriaxone. A earlier examine confirmed that tolerance quantitatively correlates with progress price [44]. In that examine, progress charges had been assorted by completely different however fixed progress environments. The protecting impact of progress inhibition could not happen instantaneously after discount of progress price explaining why gonococci weren’t protected in opposition to ceftriaxone regardless of progress inhibition.
In abstract, we reveal a transition from uncorrelated single-cell fluctuations to collective dynamics of membrane polarization in gonococcal colonies and supply robust proof that the native oxygen gradient governs this transition. Additional, we discover {that a} touring shell of hyperpolarized cells demarks the differentiation into 2 subpopulations with completely different progress charges and completely different ranges of antibiotic tolerance. In future research, it will likely be essential to search out the underlying mechanism of transient hyperpolarization. We anticipate that finding out dynamical polarization patterns in creating biofilms will present novel insights into the mechanisms of biofilm differentiation in numerous bacterial species.
Supplies and strategies
Progress media and bacterial strains
Media composition, assay preparation, and setup comply with [27]. Gonococcal base agar was constructed from 10 g/L dehydrated agar (BD Biosciences, Bedford, Massachusetts, United States of America), 5 g/L NaCl (Roth, Darmstadt, Germany), 4 g/L K2HPO4 (Roth), 1 g/L KH2PO4 (Roth), 15 g/L Proteose Peptone No. 3 (BD Biosciences), 0.5 g/L soluble starch (Sigma-Aldrich, St. Louis, MO), and supplemented with 1% IsoVitaleX (IVX): 1 g/L D-glucose (Roth), 0.1 g/L L-glutamine (Roth), 0.289 g/L L-cysteine-HCL × H2O (Roth), 1 mg/L thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/LFe(NO3) 3 (Sigma-Aldrich), 0.03 mg/L thiamine HCl (Roth), 0.13 mg/L 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/L β-nicotinamide adenine dinucleotide (Roth), and 0.1 mg/L vitamin B12 (Sigma-Aldrich). GC medium is similar to the bottom agar composition however lacks agar and starch. Employed bacterial strains (Desk i in S1 Textual content) are derivatives of pressure N. gonorrhoeae MS11.
Tradition preparation for experiments within the stream chamber and beneath static circumstances
For the microscopy experiments, we used 2 completely different setups. First, micro organism had been grown in stream chambers. This situation ensures steady stream of medium and roughly fixed provide of progress sources. Second, micro organism had been grown in static tradition. This situation was used when quick depletion of progress sources was desired or dietary supplements had been added throughout the experiment. All experiments comply with the identical preparation protocol: In a single day cultures of the respective pressure had been suspended in recent GC + IVX medium at OD600 0.1 except famous in any other case. Below addition of 1% v/v ddH2O, the obtained bacterial resolution was then incubated in a shaker at 37°C and 5% CO2 for 30 min to 1 h, which allowed colonies to first disassemble after which reform freshly.
Circulation chamber experiments: Cells had been loaded right into a microfluidic stream chamber (Ibidi Luer 0.8 mm channel peak + Ibitreat) that was beforehand coated with Poly-L-Lysine (Sigma, Cat. No. P4832, 50 μg/mL) in a single day. The stream chamber was repeatedly supplemented with medium at a temperature of 37°C utilizing a peristaltic pump (mannequin 205U; Watson Marlow, Falmouth, United Kingdom) working at 2 rpm. To visualise the membrane potential, TMRM (Sigma-Aldrich) was added to the equipped medium at a focus of 0.1 μm [33].
Static situation experiments: 300 μL of the bacterial suspension at an OD of 0.1 had been pipetted right into a μ-slide 8 Nicely (Ibidi + Ibitreat), which was coated with Poly-L-Lysine (Sigma, Cat. No. P4832, 50 μg/mL) over the earlier evening. Along with the bacterial suspension, and relying on the experiment, we added 3 μm valinomycin (Sigma-Aldrich), 5 mM sodium nitrite NaNO2 (Roth), or 20 μg/mL IPG2-AM (Ion BioSciences). Oxygen scavenger system (protocatechuic acid, PCA, and protocatechuate 3,4-dioxygenase, PCD, each from Sigma-Aldrich) preparation and focus was taken from [42].
Confocal imaging
For imaging, colonies of varied sizes and diameters had been randomly chosen and imaged utilizing an inverted microscope (Ti-E Nikon) geared up with a CSU-X1 (Yokogawa) spinning disk unit, a 100× CFI Apo Tirf goal (Nikon), an EMCCD digicam (iXon 897 X-11662, Andor), and 488 nm and 561 nm excitation lasers. If not acknowledged in any other case, colonies had been imaged at their equator as soon as each minute (acquisition time 100 ms) within the brightfield, inexperienced, and purple channel (laser energy 3% and a couple of%, respectively).
3D film of hyperpolarized shell development
3D films had been obtained by recording a number of colonies contained in the stream chamber each 5 min (pressure NG194, wt inexperienced, acquisition time: 50 ms) for a complete time interval of two h. For each imaging step, we recorded a z-stack of 10 μm with a z-spacing of 0.2 μm between every picture near the underside of the microscope coverslip. Roughly 488 nm and 561 nm excitation lasers had been set to five% and 10% depth, respectively. As well as, we acquired a brightfield picture for each time level. Lastly, 3D quantity renderings and films of hyperpolarization shell formation inside colonies had been created with NIS-Parts Ar Ver. 5.02 (Construct 1271, Nikon).
Characterization of antibiotic tolerance and fraction of lifeless cells
Colonies (wt*, NG150) had been incubated for about 120 min to permit the formation of the hyperpolarized shell inside a subset of colonies. Subsequently, colonies had been handled with medium containing kanamycin, ceftriaxone, or azithromycin at a focus akin to 10× the MIC for kanamycin and ceftriaxone (200 μg/mL and 0.04 μg/mL, respectively) and 100× the MIC for azithromycin (0.64 μg/mL) for 30 min. Below management circumstances, colonies had been handled with medium that was supplemented with an equal quantity of ddH2O as an alternative. After the remedy was stopped, colonies had been once more supplemented with medium missing any antibiotics however containing SytoX (Invitrogen) at a focus of 0.2 μm. Throughout this course of, we acquired a z-stack of every colony each 10 min. The stack lined an space of 5 μm across the equatorial aircraft of the colonies, with a z-spacing of 0.5 μm between particular person slices. Imaging was continued for 4 h past the remedy part. As well as, brightfield photos had been acquired. The fraction of lifeless cells inside colonies was then decided by monitoring the inexperienced SytoX sign (Trackmate).
Willpower of single-cell potential
The cationic dye TMRM partitions between the cytoplasm (focus cin) and the extracellular area (focus cout) relying on {the electrical} potential Vm throughout the internal membrane [4]:
(1)
Right here, R is the gasoline fixed, T is absolutely the temperature, F is the Faraday fixed, and z is the valence of the dye (z = +1). Concentrations cin,out are proportional to the TMRM fluorescence intensities inside cells and outdoors of the colonies, respectively. After loading, the TMRM sign equilibrates inside roughly 5 min [33]. To derive Vm from the fluorescence intensities, we proceeded as follows.
From the recordings, single-cell membrane potentials had been obtained by first discovering cell positions for every body by the sfGFP sign utilizing the Fiji plugin Trackmate. We then averaged the TMRM sign (purple channel) over the realm of the cells at these positions. Since TMRM molecules partition between the within and outdoors of the cell relying on the presence of detrimental fees, we moreover measured the TMRM sign distant from the colonies. For each, we subtracted the contribution of the digicam noise, which we quantified from photos within the absence of cells and TMRM. Membrane potential values had been then calculated in line with Eq (1), with T = 310Ok.
We be aware that on this examine, we’ve got not corrected for results of diffraction. In a earlier examine on the single-cell degree, we did account for this impact and measured barely (roughly 10%) extra detrimental membrane potential [33]. The distinction could also be accounted to the truth that we didn’t appropriate for diffraction right here, or to the truth that cells depolarize instantly after colony formation. Importantly, the distinction is small and doesn’t have an effect on the key conclusions of this examine.
A current examine confirmed that the cationic Nernstian dye thioflavin T (ThT) can have an effect on the expansion price of E. coli [47]. We measured the expansion price of particular person gonococcal colonies within the presence of TMRM and located λ = (0.5−0.6)h−1. That is corresponding to the speed of colonies grown beneath the identical circumstances however within the absence of TMRM [32], suggesting that TMRM doesn’t considerably have an effect on the membrane potential. Furthermore, the examine with E. coli confirmed that an efflux pump impacts the ThT fluorescence sign [47]. We can’t exclude that efflux pumps have an effect on the intracellular focus of TMRM inside gonococci, leading to underestimated absolute values of the membrane potential. Nonetheless, this systematic error wouldn’t have an effect on the conclusions of this work.
Localized progress price in entrance of and behind the hyperpolarization shell
To point out that progress charges decline after passage of the hyperpolarized shell, however not previous to it, we devised a simplified binary colony progress mannequin the place we assume that the cell progress price λ = λ0 is fixed previous to the passage of the shell and λ = 0 afterwards. We begin from the differential equation for the variety of cells Nr inside spherical shells at a distance r from the middle of mass of the spherical colony: . With Nr = ϕVr, the place ϕ is the cell quantity density of the shell and Vr its quantity, we will write the temporal change in complete colony quantity as
(R: colony radius). Utilizing
, and splitting the integral in a component behind (r < rhyper) and earlier than (r > rhyper) the hyperpolarized shell, we get hold of a relation between colony radius R and the instantaneous radial shell place rhyper
Earlier than the hyperpolarized shell kinds (t < ttrans), we count on the left-hand aspect (LHS) to be equal to zero. Afterwards, the LHS evolves because the third energy of rhyper. This relation is in good settlement with experimentally obtained values of R, rhyper, (Fig iv in S1 Textual content, 15 colonies evaluated). Moreover, heterogeneous progress charges within the colonies a number of hours after incubation are in settlement with earlier findings [32].
Response–diffusion mannequin for shell propagation dynamics
We numerically solved a 2D response–diffusion mannequin for the focus area of oxygen [45]
Right here, c ≔ c(t,x,y) is the native focus of O2 inside and outdoors of the colony (: distance to colony center-of-mass), κ(c) is the oxygen consumption price by every cell, ϱ is the native cell quantity density, and D is the diffusion coefficient of oxygen within the solvent. To account for each uptake- and diffusion-limited regimes, we used the Michaelis–Menten kind
, the place OkM is the Michaelis–Menten fixed of the oxygen uptake response, and κ0 is the higher restrict of oxygen uptake per cell. As fastened parameters, we used the Michaelis fixed decided for Bacillus licheniformis, OkM = 2.5 × 103 molecules ∙ μm−3 [48], csolvent ≡ c0 = 1.3 × 105 molecules ∙ μm−3 [33]. In bulk solvent, the diffusion coefficient of oxygen is
[49]. To account for hindered diffusion within the colony, we set the oxygen diffusivity contained in the colony to 1/tenth this worth. Lastly, the product of the maximal oxygen consumption price and the mobile density in colonies is κ0ϱ = 45 ×105 molecules ∙ μm−3 min−1 [25,33]. The cell density was assumed to be fixed inside a circle of radius R, the place R is the radius of the colony, and nil outdoors. To imitate nutrient provide within the stream chamber, we set c = c0 for r = R(t) + 5 μm. We solved the mannequin utilizing a 2D DuFort–Frankel scheme in Matlab on a grid of dimension 128 × 128 in simulation models, akin to a window of 64 × 64 μm2 (ds = 0.5 μm), with c(t = 0,x,y) ≔ c0 (timestep Δt = 10−3 min). The preliminary colony radius was 6 μm. After 104 steps (10 min), we obtained a steady-state focus profile c(t,x,y) ≔ c(r) ≡ ceq(r). At that second, we allowed cells to proliferate with progress price λ = 0.01 min−1 [32] for 300 min and selected an satisfactory focus c* = 0.8 × c0, barely smaller than ceq(0). From that second, in areas the place the focus was decrease, the expansion price was set to zero. The colony radius developed as
, the place r(t,c*) is the gap of the iso-concentration circle to the colony center-of-mass. Moreover, we allowed the oxygen uptake price within the area r<r(t,c*) to decrease by an element ε ∈ [0,1]. To check the robustness of our system and outcomes, we assorted a number of key parameters (together with oxygen diffusivity, distance of boundary situation) and located that variations had little to no qualitative impression on the outcomes.
An analytical steady-state resolution to the response–diffusion equation when it comes to a Maclaurin collection was derived in [50]. To quadratic order within the center-of-mass distance r, the answer reads , the place cc is the (time-dependent) oxygen focus on the heart of the colony. From there, we get hold of the relation
beneath the idea that c0 ≈ c(R). As a result of cc is bigger than zero and can’t exceed the boundary worth c0, the vital focus c* has vary 0.5c0 < c* < c0.