Multigenerational memory and adaptive adhesion in early bacterial biofilm communities

Abstract

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Keywords: Pseudomonas aeruginosa; bacteria biofilms; cyclic AMP; surface sensing; type IV pili.

Fig. 1.

Surface-naïve and surface-sentient planktonic bacteria exhibit drastically different behavior on the surface. (A) Fluorescence reporter intensities (${\text{I}}_{\text{cAMP}}$) for WT in FC1 (Top) and FC2 (Bottom) vs. time since first exposure to bacteria ($t_n=0$). $\text{T}=29.5\hspace{0.17em}\text{h}$ represents the time when bacteria in FC1 are harvested and introduced to virgin FC2. Data points are colored by the total number of cells per heat-map bin. For clarity, overlapping points are omitted. The top left, top right, and bottom heat maps are generated from 2,104, 1,441, and 3,935 bacterium measurements every 30 s, 15 min, and 15 min, respectively. (B) Histograms of surface residence times for the tracked cells during $t_1$ = 0–20.5 h (1,365 bacteria tracked using the bright-field images), using logarithmic binning (19 logarithmically spaced bin edges between 3 s and 38 min) and plotted on log-log axes.

Fig. 2.

WT FC2 is different from WT FC1 but like the ΔpilU mutant in FC1. (A) Histograms of normalized cAMP reporter intensities (${\text{I}}_{\text{cAMP}}$) ($t_n$ = 0–1 h) using logarithmically spaced bins. Histograms are generated from n = 54, 124, and 78 measurements of bacteria in four fluorescence frames (every 15 min) for WT FC1 (T = 29.5 h), WT FC2 (T = 29.5 h), and the ΔpilU mutant in FC1. WT FC1 has a single peak centered around ${\text{I}}_{\text{cAMP}}$ ∼1.5. WT FC2 and ΔpilU FC1 have two peaks centered around ${\text{I}}_{\text{cAMP}}$ ∼1 and 4. (B) The number of surface bacteria per frame vs. time remains near zero for ∼20 h, and increases slowly for WT FC1 (Top) but increases immediately and much faster for WT FC2 (Middle) and ΔpilU FC1 (Bottom). Solid lines represent data. Dashed lines represent data fit to exponential functions. Shaded areas represent 95% CI of the fits. (C) Distribution of the total number of surface generations of the family each bacterium is part of for the same data in B. Error bars indicate relative error of $1/\sqrt{\text{no.}\hspace{0.17em}\text{of}\hspace{0.17em}\text{points}\hspace{0.17em}\text{per}\hspace{0.17em}\text{bin}}$. Bacteria in families with at least five generations are relatively rare for WT FC1 (Top) but are much more common for WT FC2 (Middle) and ΔpilU FC1 (Bottom) (vertical dashed line).

Fig. 3.

WT and surface-sensing mutants exhibit key differences in family tree architecture. (A) cAMP-dependent lacP1 promoter activity is represented as fold change of lacP1-lacZ promoter activity over a vector control. Values are reported as mean ± SD from three independent experiments with three replicates each. (***$P<0.001$ compared with WT.) (B) Proportions of “n-legged” division branching per strain, where n is the number of nondetached daughter cells postdivision. Data represent 70∼110 events per strain. χ² tests are performed to determine statistical differences in the observed proportions. n = 2 and n = 1 had χ² test P values of $1.19\times {10}^{-5}$ and $6.03\times {10}^{-6}$, respectively, indicating that there are statistical differences among the strains. Further pairwise χ² tests and the Benjamini and Yekutieli (17) procedure for controlling the false discovery rate of a family of hypothesis tests show that the ΔpilU mutant is significantly different from the other strains $\left(\ast \ast \ast \ast P<{10}^{-4}\right)$. If postdivision outcomes were randomly distributed, then the probabilities for $n=\mathrm{0,1,2}$ would be $q^2,2q\left(1-q\right),{\left(1-q\right)}^2$, respectively, where $q$ is any number between 0 and 1. There is no way to reproduce observed distributions in the figure for any value of $q$, so correlations must exist in the system. (C) Tree asymmetry $\mathrm{\lambda }$ characterizes a family’s overall division branching behavior. Circle area is proportional to the number of families with the same λ (three or more families per strain, ≥10 bacteria per family). (D) Family trees for WT and TFP-related gene deletion mutants. A small fraction of ΔpilU cells divide while vertical (open triangles), similar to Caulobacter crescentus (27).

Fig. 4.

Multigenerational cAMP–TFP signal transduction drives memory and memory loss. (A) Schematic of the stochastic model that describes multigenerational cAMP–TFP signal transduction, illustrating the model components [PilA monomer $m\left(t\right)$, cAMP signal $s\left(t\right)$, and TFP activity $A\left(t\right)$]. (Inset) The equations relating these components. (B) Comparison of model and experiments using the correlation functions $C_{AA}\left(t\right)$ (Left), $C_{ss}\left(t\right)$ (Middle), and $C_{As}\left(t\right)$ (Right) calculated from data for one family in WT FC2. Circles are experimental data. Error bars indicate relative error of $1/\sqrt{\text{no.}\hspace{0.17em}\text{of}\hspace{0.17em}\text{points}\hspace{0.17em}\text{per}\hspace{0.17em}\text{time}\hspace{0.17em}\text{lag}}$. Solid lines indicate the model fit (parameters: $\left[k,{\omega }_0,k_s,D,k_i{k}_t\right]=\left[0.12,\hspace{0.17em}0.6,\hspace{0.17em}-0.35,\hspace{0.17em}3200,\hspace{0.17em}0.025\right]$). There are negative and positive cross-correlations (Right) at time lag 0 h and ∼±5 h, respectively. (C) “Memory-loss” flow-cell experiments, where we harvest WT bacteria from FC1 at T = ∼30–40 h and maintain them in culture tubes for different time intervals ($t_r=0\hspace{0.17em}\text{h},\hspace{0.17em}14\hspace{0.17em}\text{h},\hspace{0.17em}\text{and}\hspace{0.17em}37\hspace{0.17em}\text{h}$) before introducing them to FC2. Solid lines represent data of number of surface bacteria per frame vs. time. Dashed lines represent data fit to exponential functions. Shaded areas represent 95% CI of the fits. $t_{lag}\left(t_r=0\hspace{0.17em}\text{h}\right)=4.68\hspace{0.17em}\text{h}\hspace{0.17em}\left[4.65\hspace{0.17em}\text{h},4.72\hspace{0.17em}\text{h}\right]$, $t_{lag}\left(t_r=14\hspace{0.17em}\text{h}\right)=5.46\hspace{0.17em}\text{h}\hspace{0.17em}\left[5.44\hspace{0.17em}\text{h},5.48\hspace{0.17em}\text{h}\right]$, and $t_{lag}\left(t_r=37\hspace{0.17em}\text{h}\right)=13.41\hspace{0.17em}\text{h}\hspace{0.17em}\left[13.39\hspace{0.17em}\text{h},13.42\hspace{0.17em}\text{h}\right]$. Near-complete “memory loss” occurs by $t_r=37\hspace{0.17em}\text{h}$.

Fig. 5.

Surface-sentient state involves cAMP–TFP correlations and suppression of TFP motility. (A) Plot of time-averaged (time lag 0 h) cAMP signal vs. TFP activity per bacterium for 50 bacteria in 31 families in WT FC 1 (Left) and 95 bacteria in five families in WT FC2 (Right) (T = 29.5 h). For FC1, one symbol is used for individual bacteria. For FC2, each symbol and color represents one family, and each data point is one bacterium in that family. These two quantities are uncorrelated in FC1 (Spearman correlation: $\rho =-0.153,P=0.29$) and anticorrelated in FC2 (Spearman correlation: $\rho =-0.643,P=4.69\times {10}^{-7}$). (B) Distributions of time-averaged TFP activity for bacteria that do not detach from the surface. FC2 is statistically different (Kruskal–Wallis test P value $\ll {10}^{-5}$) with approximately threefold more cells having near-zero TFP motility. (C) Model for different stages of early biofilm growth. Stage (1): When engaging a virgin surface, essentially all attaching cells detach almost immediately and do not stay long enough to divide (Bottom). The surface population is near-zero (Top). Stage (2): Cells begin to stay on the surface for more than a generation with increased cAMP and TFP activity, but typically with one-legged division branching (Bottom). The surface cell population increases from zero to a low value, since one-legged trees do not significantly increase the population (Top). Stage (3): cAMP and TFP activity correlate into coupled oscillations, resulting in a new state characterized by suppression of TFP-mediated motility and an increasing number of cells with near-zero TFP activity. These events mediate the emergence of an exponentially increasing population of biofilm bacteria (Top), made possible by the new two-legged family tree architecture (Bottom).