Transient heterogeneity in extracellular protease production by Bacillus subtilis

Jan-Willem Veening^1,2, Oleg A Igoshin³, Robyn T Eijlander¹, Reindert Nijland¹, Leendert W Hamoen² & Oscar P Kuipers¹

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Synopsis

When environmental conditions become unfavorable, the Gram-positive model bacterium Bacillus subtilis is able to employ a number of adaptive responses, such as competence development for DNA uptake, endospore formation and the production and secretion of proteolytic enzymes (Dubnau and Lovett, 2002; Piggot and Losick, 2002; Tjalsma et al, 2004). Activation of the competence or sporulation pathway occurs only in part of the population (Cahn and Fox, 1968; Hadden and Nester, 1968; Chung et al, 1994). As there are clearly two distinguishable cell types in both cases, this phenotypic variation was described as exhibiting bistability (Fujita et al, 2005; Maamar and Dubnau, 2005; Smits et al, 2005; Veening et al, 2005).

As most gene expression experiments in the stationary growth phase have been performed on the basis of population-wide studies, little is known about the gene expression profiles of the specific subpopulations. Previous genome-wide studies on sporulating cultures might have masked non-sporulation-related gene expression and putative additional levels of heterogeneity (Fawcett et al, 2000; Eichenberger et al, 2004). To reveal the expression patterns of non-sporulating cells within isogenic sporulating cultures of B. subtilis, both a genome-wide and a single-cell approach were used. First, we developed a method to separate endospore-containing cells from vegetative cells using buoyant density gradient centrifugation. The transcriptomes of the resulting subpopulations were compared using DNA-microarray technology. This analysis revealed the occurrence of substantial heterogeneity in gene expression patterns within the isogenic B. subtilis culture. Cells either sporulate or activate a number of adaptive regulatory networks such as motility and competence development. Subsequent single-cell analyses using promoter-GFP fusions confirmed the microarray results and, surprisingly, revealed further heterogeneity within the non-sporulating subpopulation (Figure 2). Only part of the cells within the vegetative subpopulation highly and transiently expresses genes encoding the extracellular proteases Bpr (bacillopeptidase) and AprE (subtilisin), both of which are under the control of the DegU transcriptional regulator (Figure 1A). These extracellular proteases are known to act as scavenging enzymes and degrade large, complex proteins to smaller peptides, which can subsequently be taken up again as a new source of nutrients. Since these proteases and the products of their degradation activity freely disperse within the (liquid) growth medium, all cells within the clonal population are expected to benefit from their activities, indicating that B. subtilis might employ cooperative or altruistic behavior.

Figure 1

aprE activation by a logic AND gate. (A) Kinetic scheme showing the reactions used to generate the computational model. Dotted boxes show the DegU system, which includes positive feedback and the bistable sporulation switch. Thin arrows represent basal transcription reactions, whereas thick arrows represent activated levels. For simplicity of the scheme, several reactions are omitted (e.g. SinR binding/dissociation, degradation of mRNA/proteins). (B) Cells require both DegUP AND Spo0AP to activate aprE transcription.

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To obtain more mechanistic insights into how such heterogeneity is generated, we performed a series of genetic experiments and developed a quantitative mathematical model that accurately describes our (genetic) observations. Our data show that degU transcription is heterogeneous and gradually increases with time in a unimodal distribution. Autoactivation of DegU is critical in reaching high levels of aprE transcription. Furthermore, phosphorylation of Spo0A, the master sporulation transcription factor, is also required to relieve the aprE promoter from repression of a number of transcriptional regulators (e.g. AbrB and SinR). Our experimental data suggest a time-window model of how heterogeneity of aprE gene expression is generated. The temporal window for aprE expression opens when at least two conditions are satisfied. Firstly, environmental signals result in phosphorylation of Spo0A and de-repression of negative repressors such as AbrB and SinR. Because only part of the population reaches the Spo0AP levels that are required to relieve the aprE promoter, only part of the population is primed to activate aprE gene expression. Secondly, an increase in the DegU phosphorylation rate (or decrease in the dephosphorylation rate) results in a higher probability of activating the degU autostimulatory loop. Thus, only cells that have high levels of both Spo0AP and DegUP will highly express aprE (Figure 1B). The aprE expression window closes when the gradual increase of Spo0AP reaches the level required to initiate endospore formation (Fujita and Losick, 2005) and may also be affected by additional factors such as cell death or induced DegU proteolysis.

Using this information, we built a qualitative mathematical model that constitutes a logic AND circuit involving the bistable sporulation pathway and the DegU autoactivation pathway (DegU system). Our model and experimental data support the hypothesis that, in the late stationary phase, the DegU system functions with parameters where only the fully activated state is operational. However, positive feedback in the system with the potential to demonstrate bistability results in slow and stochastically variable transitions from the 'OFF' to the 'ON' state. Simulations of the integrative model yield valuable insights into how aprE population heterogeneity arises from the relatively long and variable response times within the DegU system and makes testable experimental predictions.