Molecular mechanisms involved in Bacillus subtilis biofilm formation

Summary

Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil-dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programs of cellular specialization and cell-cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis. In particular, a special emphasis is placed on summarizing the most recent discoveries in the field and integrating them into the general view of these truly sophisticated microbial communities.

Introduction

Communities of surface-associated bacteria or biofilms are now seen as the predominant mode of microbial life in multiple environments and have a severe impact in our society at different levels, such as industrial pipelines, plant-roots or the development of hard-to-treat infections (Donlan, 2002; Morris and Monier, 2003; Hall-Stoodley et al., 2004; Donlan, 2008) Biofilm-associated infections are generally hard to treat because of the ability of biofilm-encased bacteria to resist a wide variety of external insults, including antibiotic treatments (Anderson and O’Toole, 2008; Bryers, 2008). This protection is due to the action of the extracellular matrix of the biofilm, which is generally composed of exopolysaccharides (EPS), proteins and sometimes nucleic acids (Sutherland, 2001a; Sutherland, 2001c, b; Whitchurch et al., 2002; Flemming and Wingender, 2010). The extracellular matrix plays a major role in holding cells together within the biofilm while simultaneously protecting them from external insults.

A natural form of a biofilm is undoubtedly constituted by multiple microbial species (Watnick and Kolter, 2000; Stoodley et al., 2002). In these habitats, bacteria grow in close proximity and interact to compete or cooperate for space and nutrients (Nadell et al., 2009). These interactions between different bacterial species require the activation of sophisticated cell-cell communication mechanisms, such as quorum sensing, quorum quenching or the secretion of secondary metabolites, like antimicrobials or siderophores (Davies et al., 1998; Dong et al., 2001; Dong and Zhang, 2005; Lopez et al., 2009d; Lopez et al., 2009c). Because biofilms in nature house a complex mixture of species, this makes molecular genetic studies extremely difficult, and research often focuses on single-species biofilms growing in artificial laboratory conditions that are easy to control and reproduce. The most common way to generate biofilms in laboratories is the formation of surface-associated communities on submerged solid surfaces that can be visualized using a microtiter dish plates coupled with the crystal violet assay (Fletcher, 1977; O’Toole and Kolter, 1998). Another form of biofilm is represented by the formation of a structured pellicle at the air-liquid interface of standing liquid cultures (Branda et al., 2001) Additionally, colonies that grow on the surface of agar dishes are now widely recognized as a form of biofilm, as they are robust and encase microbial communities in a extracellular matrix (Branda et al., 2001). This methodology has been adapted by many laboratories to assay biofilm formation in a diverse number of bacterial species, like Bacillus subtilis (Branda et al., 2001), Pseudomonas aeruginosa (Dietrich et al., 2008), Salmonella enterica (Latasa et al., 2012) or Escherichia coli (Serra et al., 2013).

Among the studied species, Bacillus subtilis is a motile, Gram-positive bacterium widely used in studies of biofilm formation (Branda et al., 2001; Vlamakis et al., 2013). B. subtilis is able to form robust biofilms that are manifested by highly structured floating pellicles that grow on the surface of liquid cultures and colonies that grow on agar plates (Branda et al., 2001; Vlamakis et al., 2013). Many derivatives of the laboratory strain B. subtilis 168 are unable to develop highly-structured biofilms due to the accumulation of mutations during laboratory propagation, a phenomenon that is known as “domestication” (Zeigler et al., 2008; McLoon et al., 2011). In contrast to the weak biofilms formed by the laboratory strains of B. subtilis, undomesticated strains of B. subtilis are generally able to develop robust and sophisticated colony architectures. The formation of robust biofilms is an attribute that is possible to observe in in a large variety of undomesticated strains isolated form natural environments (Fig. 1). This is indicative of the importance of biofilm formation in environmental samples and possibly the survival of B. subtilis in natural conditions. The genome-sequenced undomesticated strain NCIB3610 is the usual choice regarding experiments of biofilm formation (Fig. 1). Yet, a common remark of all biofilms that are generated by undomesticated strains is the abundance of architectural features which, based on the work performed in NCIB3610 in the past decades, we know this relies on a high degree of cellular specialization and cell-cell communication within the community that follows a spatiotemporal organization pattern similar to those described for more sophisticated multicellular organisms (Vlamakis et al., 2008; Lopez et al., 2009d). In this review, we focus on recent advances in the knowledge of B. subtilis biofilm formation and by extension, in the general understanding of biofilm formation.

Figure 1. Architectural differences between biofilms from distinct B. subtilis species.

Strains were grown in MSgg agar for 72 h at 30 °C. Bacillus subtilis subspecies subtilis 3610 was isolated from Marburg (Germany). Bacillus subtilis subspecies subtilis 168 is a laboratory strain. Bacillus subtilis subspecies subtilis RO-NN-1 was isolated from the Mohave dessert (USA). Bacillus subtilis subspecies subtilis AUS198 was isolated from Salzburg (Austria). Bacillus subtilis subspecies vallismortis DV1-F3 was isolated from the Death Valley (USA). Bacillus subtilis subspecies mohavensis RO-H-1 was isolated from the Mohave dessert (USA). Bacillus subtilis subspecies spizizenii DIV-B-1 was isolated from the Death Valley (USA). Bacillus subtilis subspecies spizizenii TU-B-10 was isolated from Nefta (Tunisia). Scale bar is 3 mm.

Biofilm formation

In order to generate a biofilm, cells switch from a planktonic to a sessile state by downregulating the expression of flagellar genes and inducing the expression of genes required for matrix production, led by the reinforcement of external cues (e.g. nutrient depletion, low oxygen levels or surface adherence) (Freese, 1972; Chung et al., 1994; Cairns et al., 2013; Kolodkin-Gal et al., 2013). Sessile cells start forming chains via the repression of cell-wall hydrolases and the chains become enclosed in a self-produced extracellular matrix that provides rigidity and is required for the formation of robust biofilms (Branda et al., 2006). Biofilm expansion is mediated by the action of the motile cells and the release of surfactant molecules (Angelini et al., 2009). As the biofilm expands and matures, the production of extracellular matrix continues, and the biofilm develops extensive wrinkles. This is a consequence of spatially constrained cell death that dictates the lateral compressive forces created by the stiffness of the biofilm matrix, followed by mechanical buckling of the matrix (Asally et al., 2012; Trejo et al., 2013). Overall, wrinkling has several advantages for the microcolony. First, it increases the surface-to-volume ratio in order to provide better access of cells to oxygen (Dietrich et al., 2008; Kolodkin-Gal et al., 2013). Second, wrinkles promote the formation a complex network of liquid channels within the biofilm that facilitates the circulation of liquids (Wilking et al., 2013). Finally, the biofilm ages and aerial projections develop at the surface, which serves as preferential sites of sporulation and the spread of spores (Branda et al., 2001).

The rigid extracellular matrix that constitutes B. subtilis biofilms contains exopolysaccharides (EPS) and proteins (Branda et al., 2006). The genes responsible for the production of EPS are part of the epsA-O gene operon (eps) (Branda et al., 2001; Kearns et al., 2005; Branda et al., 2006). The molecular structure of the EPS is yet to be elucidated, however, it is known that eps-defective mutants developed flat colonies and extremely fragile pellicles (Branda et al., 2006). These mutants are still able to grow in cell chains and contain remaining extracellular material due to the presence of additional matrix components of a protein nature (Branda et al., 2006). Two secreted proteins provide structural integrity to the matrix. These are TasA and TapA, which are encoded by the three-gene operon tapA-sipW-tasA (tapA operon) (Branda et al., 2006). TasA is a functional amyloid protein (Romero et al., 2010), which is secreted into the extracellular space with the help of SipW, where it self-assembles into fibers that are anchored to the cell wall by TapA (Romero et al., 2011). A tasA-defective mutant does not form a biofilm, but their defect is not as dramatic as that of the eps-defective mutants. These mutants also produce cell chains that are not held together (Branda et al., 2006). An additional protein component of the matrix has been recently discovered. BslA is secreted during the final stages of biofilm maturation and self-assembles into a hydrophobic layer on top of the biofilm where it serves as a water-repellent barrier for the community (Kobayashi and Iwano, 2012; Hobley et al., 2013). These types of water-repellent proteins are generally referred to as hydrophobins. For instance, fungal hydrophobins confer water repellency properties to fungal spores, in a way that is similar to the water-repellent properties of BslA in protecting the bacterial community of B. subtilis (Morris et al., 2011; Hobley et al., 2013). Taken together, constituent cells are encased in an extracellular matrix composed of exopolysaccharides (EPS) and protein polymers proteins (TasA and BslA).

Biofilm disassembly

One of the most important challenges in the study of biofilm formation is finding new methodologies to disperse biofilms (Karatan and Watnick, 2009; Mirani et al., 2013). Biofilm dispersal could be beneficial in, for example, eradicating biofilm-mediated chronic infections or avoiding pipeline clogging (Karatan and Watnick, 2009; Mirani et al., 2013). Because of this, many scientists actively seek for small molecules that can effectively induce biofilm dispersal. The molecules, however, need to meet certain criteria to be considered for further applications, which includes being innocuous to humans and able to reproduce the biofilm-defective phenotypes. Yet, genetic phenotypes are stable, reproducible and often difficult to replicate using chemical inhibition, a feature that has complicated the search for biofilm dispersal agents. Indeed, B. subtilis biofilms have been used to assay biofilm dispersal. It was recently reported that various D-amino acids exogenously added to the cultures causes a dispersal of B. subtilis biofilms due to their negative effect on the organization of bacteria’s cell wall and its attachment of the extracellular matrix of the cells (Kolodkin-Gal et al., 2010). A similar effect is attributed to certain D-amino acids, like D-Serine, which can replace the D-Alanine residues from the pentapeptide that crosslinks the peptidoglycan, affecting negatively the activity of certain cell wall antibiotics, like vancomycin (Pereira et al., 2007). Nevertheless, it was recently reported that toxicity of D-amino acids occurs upon addition to B. subtilis cell cultures (Cava et al., 2011; Hochbaum et al., 2011). The molecular basis of this toxicity resides on their ability to replace their L-isomers during protein synthesis and cause a general missfunction of the proteins. The toxic effect of D-amino acids should be further explored and carefully considered when studying biofilm dispersal or cell wall synthesis. In those lines, toxicity of D-serine has not been reported so far, which suggests that distinct D-amino acids may have different properties. Another small molecule described for B. subtilis biofilm dispersal is the self-produced polyamine Norspermidine (Kolodkin-Gal et al., 2012). Norspermidine interacts specifically with the exopolysaccharide of the extracellular matrix to break down existing biofilms. Subsequently, mutants unable to produce norspermidine formed long-lived biofilms (Kolodkin-Gal et al., 2012). However, detection of norspermidine in B. subtilis biofilms resulted difficult to reproduce by other laboratories, which also raise concerns regarding the capability of B. subtilis to produce the polyamine (Hobley et al., 2014). Altogether, these studies suggest that D-amino acids and norspermidine as specific biofilm disassembly triggers need to be further clarified. Under certain conditions, these molecules can affect growth or inhibit biofilm formation (Hofer, 2014).

Cell heterogeneity within the biofilm

Cells encased in biofilms are able to differentiate into subpopulations of phenotypically distinct but genetically identical cells (Lopez et al., 2009a; Lopez and Kolter, 2010a). These are subpopulations of specialized cells that produce or respond to different signals and serve distinct purposes to the overall community to strategically distribute labor and efficiently minimize energy costs (e.g. by preventing cells to enter the energy-consuming process of sporulation) (Baty et al., 2000b; Baty et al., 2000a). The regulatory network that controls cell differentiation is rather complex, yet we can distinguish three master regulators as key players in the process of cell differentiation, DegU (Dahl et al., 1992), ComA (Kunst et al., 1994; Tran et al., 2000) and Spo0A (Gaur et al., 1986; Hahn et al., 1995). These regulators are responsible for the activation of specific gene expression cascades that ultimately lead to the differentiation of all coexisting cell types in B. subtilis biofilms (Fig. 2). Moreover, differentiation into those subpopulations is a key requirement for the formation of complex colony architecture, because depletion of one of those key regulators results in dramatic defects in colony morphology (Branda et al., 2001; Kobayashi, 2007a; Verhamme et al., 2007; Kobayashi, 2008).

Figure 2. Different cell differentiation programs coexist in B. subtilis biofilms.

Schematic representation of a thin-sectioned mature biofilm, in which distinct subpopulations of cell types coexist and exhibit different spatio-temporal distribution patterns. The different genetics programs related to cell differentiation are detailed, and the genes related specifically for each differentiation process are located within the specific frame.

In a planktonic, free-living population, all cells are motile. The expression of genes required for motility is conferred when the abovementioned regulators are in an unphosphorylated state (Amati et al., 2004; Kobayashi, 2007b; Verhamme et al., 2007; Blair et al., 2008; Guttenplan et al., 2010). When cells become sessile, the master regulators DegU, ComA or Spo0A activate in response to the presence of diverse extracellular cues, which decreases the population of motile cells and favors the differentiation of cell types. This means that the subpopulation of motile cells is only present when none of the cell differentiation programs is active and are, thus, mutually exclusive to any other specialized cell type (Vlamakis et al., 2008). In contrast to the subpopulation of motile cells, the rest of the cell types generally share differentiation programs and sometimes overlap. For instance, activation of Spo0A by phosphorylation (Spo0A~P) triggers spore formation in only a subfraction of B. subtilis cells (Stragier and Losick, 1996). It is now known that there are two distinct levels of activation for Spo0A~P. A low activation level induces matrix production and a higher level favors sporulation (Fujita et al., 2005). Then, Spo0A-ON cells become matrix producers before undergoing sporulation, and both differentiation programs overlap while transitioning from one cell type to another. Spo0A-ON cells prevent flagellar motility via activation of a molecular clutch (EpsE protein) that is encoded within the eps operon (Blair et al., 2008; Guttenplan et al., 2010). Furthermore, Spo0A-ON cells are also cannibals (Gonzalez-Pastor et al., 2003; Lopez et al., 2009b). Cannibal cells are immune to the action of two self-secreted peptide toxins (Skf and Sdp toxins) that kill their sensitive siblings. This process can be considered as cannibalism, because dead cells serve as food to delay sporulation when nutrients are scarce. Activation of ComA (ComA~P) leads to the differentiation of a subfraction of cells into naturally competent cells (Magnuson et al., 1994). These cells are capable of incorporating external DNA into their genome to ultimately increase the genetic variability within the community. Activation of ComA~P is a quorum-sensing regulated process and, thus, it occurs upon reaching a certain cell density. ComA-ON cells also express the paracrine signal surfactin, which triggers the differentiation of matrix producers (Nakano et al., 1991; Lopez et al., 2009c). Thus, ComA-ON cells also differentiate into two different specialized cell types, surfactin producers and competent cells, which overlap when transitioning from one cell type to another. Activation of DegU (DegU~P) leads to a subpopulation of cells that specialize in the secretion of exoproteases and, thus, are referred to as “miners” (Veening et al., 2008). Miners promote the degradation of large biopolymers into smaller and more nutritive peptides for the community. This subpopulation differentiates preferentially from motile cells or matrix-producing cells that are located in close proximity to the air surface (Marlow et al., 2014). This is consistent with the fact that DegU-ON cells positively regulate the expression of BslA, a water-repellent protein on the surface of the biofilm that is required for biofilm integrity (Kobayashi and Iwano, 2012; Hobley et al., 2013).

The analysis of the spatial localization of these subpopulations in mature biofilms using fluorescent reporters suggested that motile cells positioned on the bottom layer of the biofilm contribute to the expansion of the community, whereas matrix producers localized in the core primarily produce the extracellular matrix and maintain its rigidity (Vlamakis et al., 2008). Spores are present on aerial structures in the top of the biofilm that facilitate their dispersion (Vlamakis et al., 2008). DegU-ON cells preferentially localize on the surface of the agar-attached biofilm, which favors the formation of the BslA layer that covers the biofilm (Marlow et al., 2014). The spatio-temporal distribution pattern of competent cells and surfactin producers is still unknown, due to the small size of this subpopulation, which prevents their detection in the overall communities.

Triggers feeding into the biofilm’s regulatory network

Differentiation of distinct subpopulation of cell types is a crucial requirement for formation of architecturally complex microbial aggregates. The activation of the different genetic programs that direct cell differentiation in B. subtilis biofilms is generally driven the action of exogenous or endogenous cues and signals. In this section, we present an overview of the known signals that feed into the cell differentiation programs and the mechanisms of action of these signals to activate the phosphorylation of the responsible master regulators Spo0A, DegU and ComA that leads differentiation of distinct subpopulation of cell types.

Activation of Spo0A requires the phosphorylative action of five different kinases (KinA-E) that are responsible for transferring a phosphoryl group to Spo0A via a phosphorelay system (LeDeaux et al., 1995; Jiang et al., 2000). Spo0A~P induces the expression of the SinI repressor, which binds to and inhibits the SinR repressor to ultimately inhibit the expression of the matrix-related genes (Kearns et al., 2005; Chai et al., 2008). The complexity of this genetic cascade prevents a comprehensive description in this review, which can be found in (Vlamakis et al., 2013). Yet, we extracted from it two interesting regulatory networks worth mentioning. First, is the SinR activity, which is antagonized by a second repressor SlrR. The SinR-SlrR complex titrates SinR and prevents SinR from repressing the expression of slrR in a double-negative feedback loop (Chu et al., 2008; Kobayashi, 2008). Second, Spo0A~P inhibits the expression of a second repressor regulatory protein AbrB (Strauch et al., 1990). This is an alternative repressor to fine tune matrix-related gene expression. AbrB acts in coordination with an AbrB-homolog repressor protein termed Abh and an AbrB inhibitory protein that is referred to as AbbA (Strauch et al., 2007; Banse et al., 2008).

The activation of KinA-E kinases is regulated by the action of specific signals. Some of these signals have been identified. KinC induces low levels of Spo0A~P when sensing the evacuation of potassium cations caused by the pores that surfactin generates in the membrane of B. subtilis (Lopez et al., 2009d). KinD senses glycerol, manganese and L-malic acid products released by plant roots (Beauregard et al., 2013; Shemesh and Chai, 2013). It also acts as a checkpoint to sense production of extracellular polysaccharides to allow cells to undergo sporulation when a critical threshold is reached (Aguilar et al., 2010). KinA and KinB were initially described to induce sporulation but new data suggest their contribution to biofilm formation. KinB acts in a complex with the respiratory apparatus and is activated when electron transport is impaired, due to certain environmental stresses (e.g., low oxygen or high iron). KinA also contributes to this process by sensing NAD+/NADH levels in the cytoplasm through direct binding to NAD+ (Kolodkin-Gal et al., 2013).

Upon phosphorylation, DegU-P can act both as an inhibitor and an activator of gene expression depending on its phosphorylation status (Verhamme et al., 2007; Murray et al., 2009). Some studies correlate the activation of DegU~P with changes in osmolarity or upon sensing specific peptidic signals (Ogura et al., 2003). One can easily reckon that the stimuli that trigger DegU are rather unknown, yet two interesting cues have been recently discovered that we want to briefly describe. It has been recently shown that the activation of DegU~P occurs upon restriction of flagellum rotation, probably as a mechanism to ensure the activation of DegU-P exclusively in cells that are attached to a surface (Hsueh et al., 2011; Cairns et al., 2013). Also, it was recently demonstrated that B. subtilis cells are able to specifically degrade the phosphorylated form of DegU-P by ClpC-mediated proteolysis (Ogura and Tsukahara, 2010).

The activation of ComA is possibly one of the best known and largely reviewed signaling cascades in B. subtilis. Because of this, we did not expand this section despite the importance of this signaling cascade. Briefly, ComA-P activates when the pheromone ComX binds to and activates the ComP sensor kinase, responsible for ComA phosphorylation (Magnuson et al., 1994). Once activated, this cascade is responsible for inducing natural competence and triggers the pathway for surfactin production (Cosby et al., 1998). ComX is a quorum-sensing signal that shows great polymorphism and is accurately recognized by each strain and rejected by other variants (Tortosa et al., 2001). Strain specificity is compensated by the production of second signal CSF (competence and sporulation factor), which acts as a species-specific signaling molecule in mediating communication between different strains (Pottathil et al., 2008).

The abovementioned genetic cascades are negatively controlled by a family of Rap phosphatases (RapA through RapK aspartyle-phosphate phosphatates) that specifically desphosphorylate and, thus, inhibit the action of the three master regulators. Each Rap phosphatate binds a specific, cognate extracellular peptide (Phr peptides), which inhibits the Rap phosphatase activity upon peptide internalization and binding (Perego et al., 1996; Perego and Hoch, 1996; Pottathil and Lazazzera, 2003). It is known that RapGH dephosphorylates DegU~P, while RapABEJ acts on the Spo0A~P phosphorelay and RapCFGHK possibly dephosphorylates ComA~P. This is an alternative cell-cell communication signaling program with a direct input in the cell differentiation programs of B. subtilis (Veening et al., 2005). This being said, the whole network of Phr-Rap regulation in B. subtilis is far from being fully understood due, in part, to the complexity of these regulatory programs, which often involves functional redundancies between the different Rap-dependent genetic cascades.

To add to this complexity, we recently discovered an additional level of complexity in the regulation of signaling networks in B. subtilis. The membrane of this bacterium (and probably all bacteria) organizes microdomains whose integrity is essential for the activation of KinC and, thus, the differentiation of SpoA-ON cells (Lopez and Kolter, 2010b). These microdomains also contain the membrane-bound protease FtsH that selectively degrades specific Rap phosphatases (Yepes et al., 2012; Bach and Bramkamp, 2013; Mielich-Suss et al., 2013). Hence, the integrity of membrane microdomains in B. subtilis membranes is important to induce biofilm formation via stabilization of KinC and FtsH. Consequently, the perturbation of the membrane microdomains using compounds like the zaragozic acid inhibits the signaling cascade to biofilm formation in B. subtillis (Lopez and Kolter, 2010b). Similarly, the protease activity of the FtsH can be inhibited by the action of the small peptide SpoVM (Cutting et al., 1997; Ramamurthi et al., 2006) and can be used as a biofilm inhibitor when synthetically synthesized and added to exponentially growing cultures (Yepes et al., 2012)

Bacillus subtilis biofilms in natural habitats

B. subtilis is traditionally considered as a soil-dwelling organism and can be found preferentially in association with plant roots in the upper rhizosphere (Pandey and Palni, 1997; Fall et al., 2004; Walker et al., 2004; Cazorla et al., 2007). Then, a central question arises as to whether the lifestyle of B. subtilis in its natural habitat entitles biofilm formation and if so, whether those biofilms are structurally similar to the ones we are currently studying in the laboratory (Costerton et al., 1987). It has been recently shown that B. subtilis colonizes plant roots, as well as plant leaves in a biofilm-dependent manner and the presence of the biofilms increases protection of the plants form a variety of pathogenic insults (Chen et al., 2012; Chen et al., 2013; Garcia-Gutierrez et al., 2013; Zeriouh et al., 2013). Importantly, the regulatory networks and structural components of the biofilms unraveled in laboratories are now proven important for the development of plant-associated biofilms. Interestingly, a recent study showed that the ability of forming a robust biofilm on plant-roots correlates with the protection towards plant-disease development (Chen et al., 2013). Moreover, the relationship turned out to be even more intimate, since plants favor biofilm colonization of the roots with the release of specific molecules (Beauregard et al., 2013; Chen et al., 2013). This is an extremely appealing result that reassures the laboratory experimentation that has been performed in the past years and it motivates innovative research that is closer to the natural habitat of B. subtilis biofilms, which are possibly the plant roots and the importance of the robust architecture of B. subtilis biofilms to attach to plant roots.

Concluding remarks

Bacillus subtilis is an exceptional model organism to study biofilm formation. In addition to its genetic tractability, B. subtilis biofilms show remarkable architectural features, which rely on complex genetic programs of cellular specialization and cell-cell communication that follow a particular spatiotemporal distribution pattern and resemble other sophisticated developmental programs from more complex organisms, like metazoans. Moreover, the current investigations of B. subtilis biofilms also provide two outstanding issues to the general scientific community interested in other areas of research, in addition to biofilm formation. First, the robustness of the B. subtilis biofilm formation assay has led to the discovery of several new regulatory pathways exhibiting sophisticated mechanisms of genetic regulation that were, so far, unprecedented in bacteria. As an example, we indeed highlight the discovery of the membrane signaling platforms in bacteria, equivalent to the lipid rafts of eukaryotic cells (Lopez and Kolter, 2010b). Second, the investigations focused on understanding the functionality of the architectural components that integrate B. subtilis biofilms continuously reveal new and unexpected components with remarkable functionalities. We would like to highlight the discovery of the protein BslA, a self-assembly hydrophobic protein with surface-active properties that allows the formation of a water-repellent coat that protects B. subtilis from external insults (Kobayashi and Iwano, 2012; Hobley et al., 2013). More extraordinary findings will surely be reported related to the ongoing research on B. subtilis biofilm formation.