Olga Lamprecht,Maryia Ratnikava,Paulina Jacek,Eugen Kaganovitch,Nina Buettner,Kirstin Fritz,Ina Biazruchka,Robin Köhler,Julian Pietsch,Victor Sourjik
Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli
Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.
Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Curli amyloid fibers are the key component of the extracellular matrix produced during biofilm formation by Escherichia coli, Salmonella enterica, and other Enterobacteriaceae [1–9]. In E. coli and S. enterica serovar Typhimurium, curli genes are organized in two divergently transcribed csgBAC and csgDEFG operons that share a common intergenic regulatory region [10]. Expression of these operons is under regulation by the stationary phase sigma factor σS (RpoS), and it thus becomes activated during the entry into the stationary phase of growth [4, 11–14]. This activation is achieved by the σS-dependent induction of the transcriptional regulator CsgD, which then controls the expression of the csgBAC operon that encodes the major curli subunit CsgA along with the curli nucleator CsgB and the chaperone CsgC [7, 8, 15]. In turn, csgD expression in E. coli and S. Typhimurium is either directly or indirectly regulated by multiple cellular factors that mediate responses to diverse environmental changes, including both global and specific transcriptional regulators, small regulatory RNAs and second messengers (reviewed in [16–19]). One of the key regulators of csgD is the transcription factor MlrA [13, 14, 20, 21]. The activity of MlrA depends on cellular levels of bacterial second messenger bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP), and in E. coli this control is known to be mediated by a pair of the interacting diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes, DgcM and PdeR, that form a ternary complex with MlrA [12, 14, 22]. MlrA is kept inactive by binding PdeR, and this interaction is relieved when the latter becomes active as a PDE thus acting as the trigger enzyme [22, 23]. This inhibition is counteracted both by DgcM, which locally produces c-di-GMP to engage PdeR, as well as by the global pool of c-di-GMP. Besides its enzymatic activity, DgcM might also activate MlrA through direct protein interaction. Another DGC/PDH pair, DgcE and PdeH, provides global regulatory input into the local DgcM-PdeR-MlrA regulation [12, 24]. At least under typical conditions used to study E. coli biofilms, this regulatory network appears to be the only c-di-GMP-dependent input that controls csgD expression [24]. Previous studies of E. coli macrocolony biofilms formed on agar plates showed that curli expression occurs in the upper layer of the colony, but even in this layer its expression remained heterogeneous [25–27], indicating an interplay between global regulation of curli gene expression by microenvironmental gradients within biofilms and its inherent stochasticity. Differentiation of E. coli into distinct subpopulations of cells either expressing or not expressing curli was also observed in submerged biofilms formed in liquid cultures, whereby curli expression was associated with cellular aggregation [28]. Furthermore, bi- or multimodality of csgD reporter activity was reported in the early stationary phase among planktonic cells in S. Typhimurium [29, 30] and E. coli [27]. Given established c-di-GMP-dependent regulation of CsgD activity, it was proposed that bistable curli expression originates from a toggle switch created by mutual inhibition between DgcM and PdeR, which could act as a bistable switch on csgD gene expression [27, 31]. In this study we demonstrate that differentiation of E. coli csgBAC operon expression, leading to formation of distinct subpopulations of curli-positive and -negative cells, occurs not only in submerged and macrocolony biofilms but also stochastically in a well-stirred planktonic culture and thus in the absence of any environmental gradients. Similar stochastic and reversible differentiation could further be observed among cells growing in conditioned medium in the microfluidic device. Although the c-di-GMP regulatory network consisting of the DgcE/PdeH and DgcM/PdeR pairs modulates the fraction of curli-positive cells in the population and the dynamics and uniformity of curli gene activation, it is not required to establish the bimodality of expression.

All strains and plasmids used in this study are listed in S1 Table. A derivative of E. coli W3110 [26] that was engineered to encode a chromosomal transcriptional sfGFP reporter downstream of the csgA gene [28] (VS1146) was used here as the wild-type strain. Gene deletions were obtained with the help of P1 phage transduction using strains of the Keio collection [32] as donors, and the kanamycin resistance cassette was removed using FLP recombinase [33]. For gene expression, dgcE and pdeH genes were cloned into the pTrc99A vector [34].

Planktonic E. coli cultures were grown in tryptone broth (TB) medium (10 g tryptone, 5 g NaCl per liter, pH 7.0) or lysogeny broth (LB) medium without salt (10 g tryptone, 5 g yeast extract, per liter, pH 7.0), supplemented with antibiotics where necessary. Overnight cultures were inoculated from LB agar plates, grown at 30°C and diluted 1:100, unless indicated otherwise, in 5–10 ml of fresh TB and grown at 30°C at 200 rpm in 100 ml flasks in a rotary shaker until the indicated OD600 or overnight (18–25 h; OD600 ~ 1.3–1.8). Alternatively, cultures were grown in TB in 96-well plates in a plate reader, with 200 μl culture per well, using cycles of 10 mins orbital shaking and 20 mins without shaking, except data in Fig 1A were alternating ~10 mins cycles of orbital and linear shaking were used instead to improve mixing. Where indicated, bacterial cultures were supplemented with either 1 mM L-serine (after 6 h of growth) or 0.1–10 mg/l DL-serine hydroxamate at inoculation.

Submerged biofilms were grown and quantified as described previously [28], with minor modifications. Overnight bacterial cultures grown in TB were diluted 1:100 in fresh TB medium and grown at 200 rpm and 30°C in a rotary shaker to OD600 of 0.5. The cultures were then diluted in fresh TB medium to a final OD600 of 0.05, and 300 μl was loaded into a 96-well plate (Corning Costar, flat bottom; Sigma-Aldrich, Germany) and incubated without shaking at 30°C for 46 h. For quantification of biofilm formation, the non-attached cells were removed and the wells were washed once with phosphate-buffered saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4). Attached cells were fixed for 20 min with 300 μl of 96% ethanol, allowed to dry for 40 min, and stained with 300 μl of 0.1% crystal violet (CV) solution for 15 min at room temperature. The wells were subsequently washed twice with 1x PBS, incubated with 300 μl of 96% ethanol for 35 min and the CV absorption was measured at OD595 using INFINITE 200 PROplate reader (Tecan Group Ltd., Switzerland). Obtained CV values were normalized to the OD600 values of the respective biofilm cultures.

Macrocolony biofilms were grown as described previously [26]. Briefly, 5 μl of the overnight liquid culture grown at 37°C in LB medium (10 g tryptone, 5 g NaCl, and 5 g yeast extract per liter) was spotted on salt-free LB agar plates supplemented with Congo red (40 μg/ml). Plates were incubated at 28°C for 8 days.

Measurements of GFP expression in an INFINITE M1000 PRO plate reader (Tecan Group Ltd., Switzerland) were done using fluorescence excitation at 483 nm and emission at 535 nm. Relative fluorescence was calculated by normalizing to corresponding OD600 values of the culture. For fluorescence measurements using flow cytometry, aliquots of 40–300 μl of liquid bacterial cultures were mixed with 2 ml of tethering buffer (10 mM KH2PO4, 10 mM K2HPO4, 0.1 mM EDTA, 1 μM L-methionine, 10 mM lactic acid, pH 7.0). Macrocolonies were collected from the plate, resuspended in 10 ml of tethering buffer and then aliquots of 40 μl were mixed with 2 ml of fresh tethering buffer. All samples were vigorously vortexed and then immediately subjected to flow cytometric analysis using BD LSRFortessa Sorp cell analyzer (BD Biosciences, Germany) using 488-nm laser. In each experimental run, 50,000 individual cells were analyzed. Absence of cell aggregation was confirmed by using forward scatter (FSC) and side scatter (SSC) parameters. Data were analyzed using FlowJo software version v10.7.1 (FlowJo LLC, Ashland, OR, US).

Conditioned medium was prepared by cultivating wild-type E. coli in TB in a rotary shaker at 30°C for 20 h, after which the cell suspension was centrifuged at 4000 rpm for 10 min, medium was filter-sterilized and stored at 4°C. Mother machine [35] microfluidics devices were fabricated using a two-layer soft lithography [36]. E. coli cells from the overnight culture in TB were loaded into the mother machine by manual infusion of the cell suspension through one of the two inlets using a 1-ml syringe. Cells were first allowed to grow at 30°C for 4 h in fresh TB, then switched to the conditioned TB and cultivated for up to 26 h. Phase contrast and GFP fluorescence images were acquired using a Nikon Eclipse Ti-E inverted microscope with a time interval of 10 min. Cell segmentation was performed on phase contrast images using of a fully convolutional neural network based on the U-net architecture [37]. Details of microfluidics experiments and image analysis are described in S1, S2 and S3 Protocols.

In order to characterize curli expression in planktonic culture of E. coli, we followed the induction of a chromosomal transcriptional reporter for the csgBAC operon, where the gene encoding stable green fluorescent protein (GFP) was cloned downstream of csgA with a strong ribosome binding site as part of the same polycistronic RNA [28]. In a previous study of submerged E. coli biofilms, this reporter showed bimodal expression both in the surface-attached biofilm and in the pellicle at the liquid-air interface, and its activity correlated with the recruitment of individual cells into multicellular aggregates [28]. When E. coli culture was grown at 30°C in tryptone broth (TB) liquid medium with agitation, this reporter became induced during the transition to stationary phase (Fig 1A), which is consistent with previous reports [12, 14, 27]. The GFP reporter level remains high in the stationary phase, which also explains its initially high fluorescence in our plate reader experiments, where the culture was inoculated using stationary-phase cells. The observed induction of curli expression occurred at similar densities in the cultures with different initial inoculum size. The onset of induction apparently coincided with the reduction of the growth rate, which might occur due to depletion of amino acids in the medium and induction of the stringent response [38, 39], consistent with the proposed role of the stringent response in the regulatory cascade leading to curli gene expression [18, 23]. In agreement with that, curli expression was strongly reduced when E. coli cultures were grown in a concentrated TB medium (S1A Fig) or when TB medium was supplemented with serine (S2A Fig). Moreover, curli reporter induction was enhanced by the addition of serine hydroxamate (SHX), which is known to mimic amino acid starvation and induce the stringent response [40] (S2A Fig). In order to investigate whether curli expression was uniform or heterogeneous within planktonic E. coli populations, we next measured curli reporter activity in individual cells using flow cytometry. The reporter was induced only in a fraction of cells, and this bimodality of curli expression became increasingly more pronounced at later stages of culture growth, reaching its maximum in the overnight culture (Fig 1B). Thus, the bimodal induction of curli gene expression is observed not only in biofilms but also in well-mixed planktonic cultures. Upon inspection of the flask culture by microscopy, no cell aggregation could be observed, consistent with weakness of curli-mediated interactions [27] and suggesting that curli gene induction in a subpopulation of bacteria is not induced by the formation of suspended biofilm-like aggregates [41]. While curli activation was more pronounced in a cell culture growing in an orbital shaker in a flask (Fig 1B), bimodality was also observed for cultures grown in a plate reader (S1B and S2B Figs). Notably, stimulation of curli expression by SHX, or its suppression by additional nutrients, affected the fraction of positive cells rather than their expression levels (S1B and S2B Figs).

Subsequently, we investigated dependence of bimodal activation of curli gene expression in planktonic culture on the known regulation of curli expression in E. coli by the global (DgcE and PdeH) and local (DgcM and PdeR) pairs of DGC/PDE enzymes [12, 14, 22] (Fig 2A). In agreement with this model of c-di-GMP-dependent regulation, activation of curli reporter was completely abolished in the absence of MlrA (Fig 2B), and it was strongly reduced by deletions of dgcE and dgcM and enhanced by deletions of pdeH and pdeR genes (Fig 2C). Notably, under these conditions a small but discernible fraction of curli-positive cells (~6%) could still be detected in cultures of both DGC gene deletion strains, and a small fraction of curli-negative cells (1–3%) was observed for both PDE gene deletion strains, indicating that deletions of these genes do not eliminate the bimodality of curli gene expression or its levels in curli-positive cells but rather change the probability of curli genes to become activated in individual cells. Consistently, bimodality was also retained upon combined deletions of pairs of DGC/PDE genes. Removal of the entire global level of c-di-GMP regulation led to a bimodal pattern of curli reporter activation within the population of ΔpdeH ΔdgcE cells (Fig 2D) that was similar to that observed in the wild-type culture, despite a smaller fraction of curli-positive cells in the deletion strain. Even more surprisingly, the distribution of curli reporter expression remained bimodal upon removal of the local level of c-di-GMP regulation in ΔpdeR ΔdgcM strain, although the fraction of curli-positive cells was reduced in this background, too. We observed that level of curli gene expression in ΔpdeR ΔdgcM strain was lower than in ΔpdeR strain, which confirms that DgcM can promote curli expression independently of PdeR [12, 14, 22] (Fig 2A). This conclusion is further supported by the comparison between the triple deletion strain ΔpdeH ΔdgcE ΔpdeR that expresses only DgcM (Fig 2E) and the quadruple deletion strain ΔpdeH ΔdgcE ΔpdeR ΔdgcM that lacks the entire regulatory network (Fig 2F), with much higher fraction of curli-positive cells observed in the former background. Most importantly, the bimodality of curli expression was still observed in the quadruple deletion strain (Fig 2F). We thus conclude that, whereas the known c-di-GMP-dependent regulation of MlrA activity does clearly affect the fraction of curli-positive cells, it is not required to establish bimodality of curli gene expression in the cell population. The network has further only little impact on the level of curli reporter activity in individual positive cells (i.e., on position of the positive peak in the flow cytometry data), although reporter intensity in positive cells appeared to be slightly reduced in the strains lacking DgcM. Vibrio cholerae transcriptional regulator VpsT, a close homologue of CsgD, has been shown to be directly regulated by binding to c-di-GMP [42]. Furthermore, in both S. Typhimurium and E. coli, curli gene expression might also be regulated by c-di-GMP independently of csgD transcription [43–45]. We thus aimed to examine whether E. coli curli gene expression was no longer sensitive to the global cellular level of c-di-GMP in the absence of the local PdeR/DgcM regulatory module. Indeed, whereas the overexpression of DgcE or of PdeH, which both affect the global pool of c-di-GMP in E. coli, had strong impacts on the fraction of curli-positive cells in the wild type, the quadruple mutant was insensitive to such overexpression (Fig 3). This result suggests that in this background, the expression of the csgBAC reporter is indeed no longer affected by global c-di-GMP levels. This further excludes regulation by other DGCs that contribute to the common c-di-GMP pool, although it does not rule out hypothetical regulation by a local DGC/PDE pair that is insensitive to global c-di-GMP levels.

We next explored how the fraction of curli-positive cells in the population depends on the conditions of culture growth, with or without regulation by c-di-GMP. As noted above, when the wild-type E. coli culture was grown in multi-well plates, both the fraction of curli-positive cells and the level of reporter activity in positive cells were moderately reduced compared to incubation in a flask in an orbital shaker (Figs 2B, 4A and S1B). The reduction in the number of curli-positive cells under these growth conditions was even more pronounced for ΔpdeR ΔdgcM or ΔpdeH ΔdgcE ΔpdeR ΔdgcM strains (Figs 4A and S3), where only a small fraction of cells (12%) became positive, compared to 40–50% in the flask culture (Fig 2D and 2F). Interestingly, this difference was less for the ΔpdeH ΔdgcE strain (24% vs 30%). Deletions of individual DGC or PDE genes generally showed expected phenotypes, but fractions of curli-positive cells in ΔpdeH and ΔpdeR strains were again reduced compared with flask cultures (Fig 2C). Another notable difference between flask and multi-well plate cultures was that the low-fluorescence peak of the wild-type culture was not fully negative but apparently contained a large fraction of cells with incompletely activated curli reporter, which could also be seen in the ΔpdeH or ΔpdeR strains but not in the ΔpdeH ΔdgcE ΔpdeR ΔdgcM, ΔpdeR ΔdgcM or ΔpdeH ΔdgcE strains. Similar results were obtained even upon prolonged incubation in the plate reader (S4 Fig), confirming that the observed difference with the overnight flask culture was not because of the different growth stage but rather due to differences in growth conditions. We further tested reporter activation under growth conditions that favor biofilm formation. During formation of static submerged biofilms in multi-well plates, where cultures are grown without shaking, the overall curli activation pattern in cell populations of individual or double and quadruple deletion strains (Fig 4B) was comparable to that in the flask culture grown in the orbital shaker (Fig 2B–2D and 2F), although effects of DGC and PDE gene deletions on the fraction of curli-positive cells were apparently reduced. Curli gene activation in individual mutant strains correlated well with the levels of submerged biofilm formation (S5 Fig), but the lack of regulation by c-di-GMP resulted in stronger reduction of the biofilm biomass than simply expected from its effect on curli gene expression, consistent with additional roles of c-di-GMP in biofilm formation. All strains were also grown in the form of macrocolony biofilms on agar plates containing the salt-free LB medium, as done previously [26] (Fig 4C), as well as in the liquid salt-free LB medium as a control (Fig 4D). Expression of curli reporter in individual cells within the macrocolony was analyzed by flow cytometry immediately after colony resuspension (see Materials and methods), so that the level of stable GFP reporter should reflect cell-specific curli expression within the macrocolony. The overall dependency of reporter activation on the deletions of individual DGC and PDE genes was again qualitatively similar to other growth conditions (Fig 4C). However, the fraction of curli-positive cells in the macrocolony biofilms was less sensitive to deletions of individual dgc or pdh genes than in planktonic cultures, regardless whether the latter were grown in TB (Fig 2C) or in the salt-free LB (Fig 4D). This agrees with stronger Congo red staining of macrocolonies of ΔdgcE and ΔdgcM strains compared to the ΔmlrA negative control (S6 Fig). Thus, during macrocolony biofilm formation, regulation by the network composed of DgcE/PdeH and DgcM/PdeR pairs appears to be less important for the activation of curli gene expression, possibly because other regulators contribute to this activation in a highly structured environment at the level of mlrA, csgDEFG or csgBAC operons. Importantly, the curli-positive cell fraction in both macrocolony and planktonic cultures of ΔpdeR ΔdgcM and ΔpdeH ΔdgcE ΔpdeR ΔdgcM strains growing on the salt-free LB was much larger than under other growth conditions. To summarize, under all of the tested growth conditions, regulation by the known c-di-GMP regulatory network was not required for bimodal curli activation, although it indeed affected the fraction of curli-positive cells. Nevertheless, the absence of this c-di-GMP control apparently makes the fraction of curli-positive cells in the population more sensitive to environmental conditions, since it exhibited much larger variation in the quadruple deletion compared to the wild-type strain among different growth conditions tested here.

In order to investigate the dynamics of curli gene activation, and the impact of c-di-GMP regulation, at the single-cell level, we utilized a “mother machine” device—a microfluidic chip where growth of individual bacterial cell lineages could be followed in a highly parallelized manner over multiple generations [35] (Fig 5 and S1 Protocol). Since our design of the mother machine allows rapid switching of the medium supplied to the cells (Fig 5D), we could mimic nutrient depletion in order to activate curli expression in continuously growing single cells. For that, wild-type, individual DGC or PDE, or quadruple deletion strains were first loaded into the mother machine from overnight cultures, allowed to grow in fresh TB medium for several generations, and then shifted to TB medium that was pre-conditioned by growing a batch culture (see Materials and methods and S2 Protocol). A fraction of curli-positive cells was observed at the beginning of these experiments, which was strain-specific and consistent with the expected fraction of positive cells in the overnight cultures of each respective strain. After resuming exponential growth in the fresh medium all cells turned off curli expression (Figs 6A, 6C and S7 and S1, S2 and S3 Movies and S3 Protocol). Following the shift to conditioned medium, cell growth rate was strongly reduced (Figs 6E and S8), and after several generations of slow growth, individual cells of all strains activated curli expression, while other cells remained in the curli-off state (Figs 6A, 6C, 6F and S7 and S1, S2 and S3 Movies). Importantly, although the absolute frequencies of curli-positive cells in individual mutants were somewhat different in the mother machine from those in the planktonic cultures grown in the flask (Fig 2), likely due to differences in growth conditions as observed already for different batch cultures and biofilms (Fig 4), the relative frequencies between strains were consistent, confirming that the overall control of curli gene expression by the c-di-GMP regulatory network is similar for growth in conditioned medium in the mother machine. Although the overall frequency of curli reporter activation was comparable between the wild-type and ΔpdeH ΔdgcE ΔpdeR ΔdgcM cells lacking the c-di-GMP regulatory network, we further compared the dynamics of single-cell curli activation in both backgrounds. First, we observed that, in both strains, curli reporter activation was transient—and after several generations in the curli-on state individual cells turned curli expression off again during continuous growth in the conditioned medium (Figs 6B, 6D, S8, and S9). In some cases, the initial transient pulse of activation was even followed by a second activation event. Although such transient curli reporter activation was observed in both backgrounds, with a similar delay after exposure to conditioned medium (Fig 6H), its dynamics showed several important differences between the strains. Most clearly, the rate of curli activation in individual cells was higher in the absence of the c-di-GMP regulatory network (Figs 6G, 6I, S10A and S10B), which led to stronger but more transient increase in the curli reporter fluorescence (Figs 6J, 6K and S10B). Additionally, the level of reporter induction showed greater intercellular heterogeneity in the ΔpdeH ΔdgcE ΔpdeR ΔdgcM strain (Figs 6D, 6J and S9). Thus, the control of curli expression by c-di-GMP reduces the rate but also the cell-to-cell variability of curli gene induction within population.

Expression of the curli biofilm matrix genes is known to be heterogeneous or even bistable in communities of E. coli [25–28] and S. Typhimurium [29, 30], which might have important functional consequences for the biomechanics of bacterial biofilms [27] and for stress resistance and virulence of bacterial populations [29, 30]. In the well-structured macrocolony biofilms, differentiation into subpopulations with different levels of curli matrix production is largely deterministic and driven by gradients of nutrients and oxygen [18], but bimodality of curli expression might also emerge stochastically, in a well-mixed population or between cells within the same layer of a macrocolony [27, 29, 30]. How this bimodality originates within the extremely complex regulatory network of curli genes [17, 19, 23, 27] remains a matter of debate. Generally, it is well established that the bimodality, and subsequently bistability, of gene expression in bacteria can be achieved by stochastic gene activation combined with an ultrasensitive response of the regulatory network, for example due to a positive transcriptional feedback loop or positive cooperativity [46–48]. Although earlier work proposed that bistable expression of curli might be a consequence of positive transcriptional feedback in csgD regulation [30, 49], the most recently suggested model attributed bistability to the properties of the potentially ultrasensitive c-di-GMP regulatory switch formed by DgcM, PdeR and MlrA [27, 31]. Furthermore, although this bimodal pattern of curli gene expression has typically been referred to as bistable, temporal dynamics and stability of its induction in individual cells has not been directly investigated. Here, we studied the expression of the major curli csgBAC operon in well-stirred planktonic E. coli cultures, as well as in submerged and macrocolony biofilms, and during growth in a microfluidics device. Consistent with previous studies [12, 14, 27], the induction of curli gene expression in growing E. coli cell populations was observed during entry into the stationary phase of growth. Curli activation was apparently dependent on depletion of nutrients, most likely amino acids, since it could be suppressed by increasing the levels of nutrients or, more specifically, by addition of serine. Conversely, it could be enhanced by the SHX-mediated stimulation of the stringent response, which mimics amino acid starvation. We further observed that activation of the csgBAC operon exhibited strongly pronounced bimodality under all tested conditions, resulting in two distinct, curli-positive and -negative, subpopulations of cells even in a well-stirred planktonic culture. Stochastic bimodality of curli reporter activation was confirmed in a microfluidic device, where, following shift to conditioned medium, only a fraction of the cell population turned on curli gene expression. Such differentiation is apparently consistent with previous reports of bimodal csgD expression in the stationary phase of S. Typhimurium [29, 30] and E. coli [27] culture growth. However, in contrast to the previous interpretation of such a csgD expression pattern as bistability, we observed that activation of curli gene expression during continuous cell growth in the microfluidic device was only transient and unstable. Moreover, our data indicate that the bimodality of the csgBAC expression is unlikely to be (fully) explained by the bimodal expression of csgD. Firstly, compared to the bimodality of the csgBAC reporter, the previously reported bimodality of csgD expression in E. coli [27] was much less pronounced and became apparent only in later stages of planktonic culture growth. Moreover, in that study, all wild-type cells showed activation of the csgD reporter, differing only in the level of this activation, which itself cannot explain the on/off bimodality of the csgBAC reporter activation. Secondly, and most importantly, whereas the bimodality of csgD expression was observed to be dependent on regulation by the DgcE/PdeH/DgcM/PdeR network [27], this was clearly not the case for csgBAC reporter expression. Under all tested conditions, including planktonic cultures, submerged and macrocolony biofilms, and growth in the microfluidic device, the differentiation into distinct subpopulations of positive and negative cells in our experiments occurred in the absence of this entire c-di-GMP regulatory network. Since removal of the DgcM/PdeR module further made curli gene expression insensitive to global levels of c-di-GMP, contributions of other E. coli DGCs and PDEs to curli regulation in this deletion background are unlikely, even if these enzymes might in principle affect curli gene expression in wild-type E. coli cells via the common c-di-GMP pool. We therefore conclude that c-di-GMP control might be dispensable for activation or bimodality of curli gene expression in E. coli, although we could not rule out regulation of curli gene expression by a hypothetical local DGC/PDE module that is insensitive to global c-di-GMP levels. Nevertheless, c-di-GMP control clearly plays an important role during the establishment of bimodality. Consistent with previous studies, we observed that the DGC and PDE proteins determine the fraction of curli-positive cells [22, 23, 27], although they have no or only little effect on the average level of csgBAC expression in individual cells. Our results further suggest that the DgcE/PdeH/DgcM/PdeR regulatory network might make curli gene expression in E. coli populations more robust, helping to ensure that the fraction of curli-positive cells is less variable under different culture growth conditions. This might be related to the observed effect of this network on the temporal expression dynamics in a continuously growing culture, with faster but more heterogeneous activation of curli gene expression in the absence of the c-di-GMP control, although whether and how there two different kinds of variability are connected remains to be investigated. It is further possible that the increased robustness provided by c-di-GMP regulation is due to the network-induced bimodality of csgD expression discussed above [27], which might provide an additional stabilizing feedback. How might the observed pulsatile activation of the curli-positive state originate at the single-cell level and lead to the differentiation into distinct subpopulations in E. coli culture? Pulsing in expression was proposed to be common to many gene regulatory circuits [50–52]. It was recently described in E. coli for the upstream regulator of curli, σS [53], as well as for the flagellar regulon [54, 55] that is anti-regulated with curli [28]. However, in neither of these cases did pulsing lead to bimodality of expression, and their relation to the observed pulses in curli expression thus remains to be seen. As mentioned above, in addition to stochastic pulsing, the emergence of bimodality requires an ultrasensitive network response, which could be achieved through one or several positive transcriptional feedbacks. Given the dependence of csgBAC operon expression on MlrA and CsgD, such feedback is likely to involve these two transcription factors, and possibly other regulators of mlrA, csgDEFG or csgBAC operons. Regardless of the origin of pulsatile expression, the timing and duration of curli activation pulses can explain how such transient activation leads to differentiation into two distinct subpopulations in the planktonic culture. Since the curli expression is only turned on during transition to the stationary phase, individual cells stochastically activate curli genes just before the culture growth ceases, thus making subsequent reversion to the curli-negative state impossible. Although curli gene regulation in biofilms might be more complex, spatial stratification of gene expression in the macrocolony biofilms was proposed to resemble the temporal regulation of expression in planktonic cultures [18, 26]. Thus, also in this case, curli activation would happen in a cell layer just before it ceases to grow, fixing the differentiation between curli-positive and curli-negative cells within this colony layer.

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