Short Description

Peptidosomes can be a powerful co-culture technique to physically separate bacterial populations without limiting their ability to communicate with each other by exchanging signalling molecules. This part of EncaBcillus is focused on proofing the concept of communication between encapsulated bacteria by using the regulatory system for competence development in Bacillus subtilis which is based on quorum sensing.

Background

In order to solve complex tasks, it is reasonable to break down a problem into small subunits that can be addressed by specialists individually. This concept is widely implemented in modern economy but can also be applied to synthetic biology [1]. In nature, such strategies have been evolved over millions of years. A good example is the formation of biofilms by the gram-positive bacterium Bacillus subtilis (B. subtilis). Here, cell-cell interactions lead to cellular differentiation processes and thus enables an effective division of labour [2]. However, the development of synthetic consortiums consisting of several different bacterial strains, with each executing distinct tasks remains challenging [3].

By encapsulating subpopulations within molecular cages, called Peptidosomes, we want to physically separate co-working bacteria while still assuring their cooperativity to fulfil one major task. In this context communication between the bacteria is indispensable, thus we had to make sure that the Peptidosome membrane is permeable for small molecules and that the bacteria are still able to interact. In this part of EncaBcillus we will show that the encapsulation of one population does not limit its ability to communicate with cells in the surrounding medium.

The regulatory system for competence development in B. subtilis

The well-studied regulatory system for competence development in B. subtilis provided a genetic set-up based on quorum sensing that we used as a proof of principle [4] (figure 1). B. subtilis constantly secretes the ComX pheromone, a 9- to 10-amino acid oligopeptide, as a signalling molecule (a). By rising cell-density, the ComX-concentration in the surrounding medium increases until it reaches a threshold and activates ComP, a membrane-spanning protein kinase (b). The kinase reacts to the accumulation of ComX by phosphorylating the response regulator ComA (c) which then works as a transcription factor by binding to several promoters and enhancing their activity (d) [5,6].

The most important promoter regulated by ComA and involved in competence development is the promoter of the srfA operon. This operon contains not only genes for the production of the antibiotic surfactin (e) but also for ComS (f), another small peptide that prevents the degradation of the autoregulated transcription factor ComK (g). ComK activates expression of more than 100 genes, including comG, that is part of the transformation machinery (h) [7,8]. Consequently, B. subtilis can take up DNA from the environment either as direct nutrient source or incorporate the DNA via homologues recombination into its own genome.

Communication between encapsulated bacteria and bacteria in the surroundings

Based on this genetic background, we wanted to set-up a simple experiment that shows communication between two B. subtilis strains. The basic idea is comparable to a radio broadcast: you need a transmitter that sends out the radio waves and a receiver that receives the waves and convert them into an acoustical signal. Transferred to our project this means that we need a sender strain (SeSt) that secretes the ComX pheromone, and a receiver strain (ReSt) that reacts to the ComX-stimulation by producing an easy-detectable output (figure 2).

In our case, we fused target promoters of the competence machinery of B. subtilis to the lux operon and measured luminescence output. Additionally, the ReSt needed to be comX-deficient to prevent autoinduction. We encapsulated the ReSt within Peptidosomes and incubated it together with the SeSt in the surrounding medium. Thus, we expected ComX that is produced outside the Peptidosme to diffuse through the membrane and stimulate the ReSt. As a result, the luminescence signal should be restricted to the Peptidosome.

Image: Experimental setting: Peptidosome with ReSt and SeSt in surrounding medium, schematic by AnastasiaL Fig. 2: Co-culture of the receiver strain (ReSt) and the sender strain (SeSt) using Peptidosomes. The ReSt in the surrounding medium secretes the ComX pheromone and stimulates luminescence of the encapsulated SeSt via quorum sensing.

References

[1]	Goers, L., Freemont, P. and Polizzi, K.M. (2014) Co-culture systems and technologies: taking synthetic biology to the next level. J. R. Soc. Interface 11, 20140065
[2]	Mielich‐Suss, B. and Lopez, D. (2015) Molecular mechanisms involved in Bacillus subtilis biofilm formation. Environ. Microbiol. 17, 555–565
[3]	Brenner, K., You, L. and Arnold, F.H. (2008) Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol. 26, 483-489
[4]	Hamoen, L.W., Venema G. and Kuipers O.P. (2003) Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology. 149, 9-17
[5]	Wolf, D., Rippa, V., Mobarec, J.C., Sauer, P., Adlung, L., Kolb, P. and Bischofs, I.B. (2016) The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs. Nucleic Acids Res. 44(5), 2160-72
[6]	Griffith, K.L. and Grossman, A.D. (2008) A degenerate tripartite DNA-binding site required for activation of ComA-dependent quorum response gene expression in Bacillus subtilis. J Mol Biol. 381(2), 261-75
[7]	Comella, N. and Grossman, A.D. (2005) Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis. Mol Microbiol. 57(4),1159-74
[8]	Susanna, K.A., van der Werff, A.F., den Hengst, C.D., Calles, B., Salas, M., Venema, G., Hamoen, L.W. and Kuipers, O.P. (2004) Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis. J Bacteriol. 186(4), 1120-8