The Roles of Calcium Signaling and Calcium Deposition in Microbial Multicellularity

Abstract

Calcium signaling is an essential mediator of signal-controlling gene expression in most developmental systems. In addition, calcium has established extracellular functions as a structural component of biogenic minerals found in complex tissues. In bacteria, the formation of calcium carbonate structures is associated with complex colony morphology. Genes promoting the formation of biogenic minerals are essential for proper biofilm development and protection against antimicrobial solutes and toxins.

Here we review recent findings on the role of calcium and calcium signaling as emerging regulators of biofilm formation in beneficial bacteria, as well as essential mediators of biofilm formation and virulence in human pathogens.

The presented analysis concludes that the new understanding of calcium signaling may help to improve the performance of beneficial strains for sustainable agriculture, microbiome manipulation, and sustainable construction. Unraveling the roles of calcium may also promote the development of novel therapies against biofilm infections that target calcium uptake, calcium sensors, and calcium carbonate deposition.

Introduction

Calcium, the third most abundant metal in nature, was adopted as a regulator of organismal behavior at an early evolutionary stage. Calcium signaling is an essential mediator of gene expression in developmental systems. During multicellular development, calcium has established extracellular functions as a structural component of biogenic minerals. The formation of these structured minerals requires a regulated developmental process. During the maturation of these minerals, they actively interact with the organic extracellular matrix [1], [2].

In surface-associated bacterial communities called biofilms, calcium promotes the formation of hierarchically structured organic−inorganic matrices reminiscent of the calcium scaffolds generated by eukaryotic organisms, such as shells [1], bones, and teeth [2]. Biofilm formation shares many features with multicellular differentiation but was not extensively studied as calcium-dependent behavior.

Besides calcium, several metals were shown to play a fundamental role in biofilm development. For example, ferric and ferrous cations are tightly connected to microbial development. Iron acquisition by siderophores is necessary for biofilm formation in Gram-positive and Gram-negative bacteria [3, 4]. Fe is essential for cellular survival while calcium is dispensable, but the requirement of biofilm cells for Fe were shown to be significantly higher than the requirement of planktonic cells, and indicated a specific role for this Fe in biofilm formation [5].

Calcium only recently emerged as a fundamental regulator of microbial development and biofilm formation [6]. In biofilms, calcium plays similar roles to those established in multicellular eukaryotes, and regulates motility, adhesion, and gene expression [7]. Moreover, calcium mineralization and its interactions with the organic extracellular matrix can impact essential biofilm properties, including permeability and diffusivity of antibiotics and small molecule solutes, community metabolism, mechanical strength, and antimicrobial susceptibility [8]. Recent findings demonstrated the presence of intracellular calcium carbonate storages (intracellular calcium mineralization) in biofilm-forming bacteria for both photosynthetic and heterotrophic species [9, 10]. These results indicated that biofilm cells can maintain a source of intracellular calcium that can be used for its structural or/and signaling function.

This review will discuss the emerging themes of calcium deposition and signaling, their roles in microbial biofilms, and their biotechnological, ecological, and medical applications. We will discuss recent findings that suggest that the regulation of calcium expands well into the microbial domain of life, where it covers nearly all aspects of cell function and controls many processes, including the transition to multicellularity.

Calcium Carbonate and Carbonate Producing Enzymes Contribute to Biofilm Formation

In ecological scenarios, the most common biogenic minerals formed by eukaryotes are calcite and aragonite, which support the three-dimensional (3D) organization of mollusks, echinoderms, calcisponges, corals, certain algae, and others [11]. For example, corals are single-cell polyps held together by calcium carbonate scaffolds to support their resilient and complex structure [11].

The most advanced understanding of biologically regulated calcium mineralization and the formation of a functional mineral has been achieved for Bacillus subtilis, where the formation of calcium carbonate structures is associated with an increased complexity of colony morphology [12–14]. B. subtilis is a Gram-positive soil-dwelling bacterium associated with plant roots. It is commonly used as a biocontrol agent to protect plants from fungal and viral infections [15]. It can also reside in the human gastrointestinal tract and is currently widely used as a probiotic intended to promote digestive health and a healthy immune system [16].

One indication that calcium biomineralization supports the formation of 3D biofilm structure is the discovered necessity of genes canonically linked to calcium deposition for biofilm formation. For example, microbial-induced calcium carbonate precipitation can be driven by the hydrolysis of urea by urease [11, 12, 17]. The products of urea hydrolysis, carbonic acid, and ammonia increase the pH of the reaction’s local environment, promoting calcium precipitation and, thereby, calcium mineralization [11]. B. subtilis Urease mutants failed to generate complex architecture in biomineralization media and formed flat featureless biofilm colonies (highly reminiscent of colonies formed by exopolysaccharides-deficient mutants), supporting a function of calcium carbonate precipitation in biofilm development [18]. The genes encoding urease were shown to be expressed in biofilm cells under nitrogen statvation [19] as ureA transcription is activated by TnrA and repressed by GlnR [20].

The chemical inhibition of urease also reduces the development of complex morphology in Pseudomonas aeruginosa biofilms [14, 21]. This specie can cause devastating chronic biofilm infections in compromised hosts, such as in cystic fibrosis (CF) patients and those with burn wounds or implanted medical devices [22]. P. aeruginosa belongs to the ESKAPE group of pathogens, bacterial pathogens with increased virulence, persistence, and transmissibility [23] that has been recognized by both the CDC (Centers for Disease Control and Prevention) and the WHO (world health organization) as a severe threat requiring urgent development of new therapeutics.

In addition to urease, carbonic anhydrases (CAs) can facilitate calcium carbonate mineralization. These zinc-binding enzymes catalyze the reversible conversion of carbon dioxide and water to bicarbonate and one proton. Specific CAs are involved in the carbonate biomineralization in distinct metazoan lineages, including sponges [24], and their role in the microbial mineralization of calcium carbonate was also recently reported [14, 21, 25]. Consistent with the role of carbonate in the establishment of colony-biofilms, overexpression of the CA YvdA enhanced the formation of complex morphological patterns in B. subtilis colony-biofilms [13]. However, whether these patterns were associated with enhanced calcite deposition remains to be determined.

The role of CAs in the calcium-dependent shaping of the biofilms was also observed in P. aeruginosa as the depletion of three β class CAs impaired the formation of cell clusters and pattern formation in P. aeruginosa PAO1 biofilms [25]. These altered patterns are likely due to the changes in the spatial distribution of cells and their aggregates within the cell clusters caused by the deposition of extracellular calcium carbonate. Both cell aggregation and calcium deposition were dependent on the presence/activity of one of the three b-class carbonic anhydrases encoded in the PAO1 genome, psCA1, and were diminished by the treatment with CA inhibitors [25]. Calcium carbonate was also recently shown to have a role in complex structures formed by Mycobacterium smegmatis [18] and Mycobacterium abscessus [21], a soil bacterium and emerging pathogen belonging to the Actinomycetota phylum. In both Actinobacteria, the inhibition of CA and/or urease impacted biofilm colony architecture [18], [21].

Several factors and processes can impact calcium carbonate mineralization. Alkaline pH effectively promotes calcium carbonate precipitation and can occur within microenvironments within a biofilm. In addition to ureolysis, bacterial metabolic pathways that can increase the solution pH include photosynthesis, ammonification, denitrification, sulfate reduction, and formate oxidation [26, 27]. For most, if not all, of these pathways, it remains to be determined whether their local and temporal activation supports matrix mineralization.

Calcium was also shown to promote biofilm formation through the regulation of aggregation by oral pathogens from the Streptococcus mutans group [28]. The carboxyl and phosphate groups on the peptidoglycan of the cell wall are the interaction sites with calcium cations. In these model organisms, the association of calcium with bacterial cells only took place in external pH above pH 6.5, indicating that in oral plaques, calcium precipitation is probably induced rather than regulated by the microbial cells [28].

These findings extend the role of biogenic carbonate minerals in supporting structure formation in bacterial biofilms and establish that mineral matrix provides structural stability to the community. The exact functions of calcium and calcium carbonate deposits in biofilm structure are particularly interesting. The following sections will highlight the diverse mechanisms calcium regulates microbial development and physiology.

The Biofilm Extracellular Matrix interacts with Calcium and Calcium Carbonate.

The biofilm extracellular matrix was studied extensively for cell-cell and cell-substrate adhesion [29]. In multicellular organisms, the extracellular matrices absorb Ca²⁺ and promote calcium carbonate formation by providing additional nucleation sites, one of the main prerequisites of calcium mineralization [2, 30] A classic example is the maturation of amorphous calcium phosphate to a crystalline bone within the collagen matrix [2]. Whether calcium mineralization occurs within the cell (controlled/regulated biomineralization) [8] or outside the cell (MICP-Microbial Induced Calcium Precipitation [11]), the organic extracellular matrix is crucial to support mineralization and allows significant accumulation of calcium carbonate (estimated by the ratio between the dry weight of the mineral the organic biomass).

Matrix proteins:

Emerging research on calcium mineralization in B. subtilis provides direct evidence of mineral absorption and assembly supported by the extracellular matrix components secreted during biofilm formation [13, 14, 18, 31, 32]. By mapping the organic extracellular matrix (ECM) signature and the mineral signature by X-ray diffraction (XRD), Azulay et al. discovered a clear bias in the metal binding properties of the extracellular matrix. Calcium was found to be preferentially bound and retained within the biofilm, thus maintaining a high concentration gradient, and enriching for local Ca concentrations relative to the surrounding medium [32]. Other ions, such as Zn, Mn, and Fe were freer to diffuse and flow as solutes, potentially accumulating in the water channels below the wrinkles of the colonies. The relative abundance of Zn, Mn, and Fe was similar in ECM mutant biofilms and the bulk of WT biofilms (away from wrinkles), suggesting that extracellular matrix binds calcium. This study also identified a major component of B. subtilis organic matrix, TasA, that forms fibrils during interactions with hydrophobic surfaces and at low pH [16]. These fibrils are atypical as TasA monomers assembled into fibres through donor-strand exchange, and TasA β-sheet-rich fibres are not amyloids per se [33]. TasA was suggested to serve as a scaffold for binding calcium within biofilm matrix and support calcite formation: X-ray fluorescence analysis supported that TasA bundles formed in high-salt and high-protein concentrations accumulated more Ca relative to Zn, Mn, and Fe [32]. These results are compatible with previous observations that the matrix proteins TasA and TapA induced the formation of calcium carbonate crystals distinct from the abiotically precipitated calcium carbonate [13, 31, 32, 34, 35].

TasA is not the only biofilm adhesin that interacts with calcium in a manner that is strictly dependent on the biofilm microenvironment. In the Gram-positive pathogen Staphylococcus aureus, biofilm-associated proteins (Baps) are a surface proteins that mediate adhesion and biofilm development [36]. In one such protein, the predicted amyloidogenic regions B and SP (hereafter BSP, aa 361–947) form a consensus of fourteen short peptide segments. Recently, it was determined that the BSP amyloid structure and function are heavily influenced by interaction networks and mediate bacterial aggregation and biofilm formation. The BSP monomer has an asymmetric dumbbell-shaped structure that can be further divided into five modules (1–5): modules 1 (aa 361–541) and 2 (aa 542–728) constitute the N-terminal lobe, whereas modules 4 (aa 775–859) and 5 (aa 860–947) constitute the C-terminal lobe. The N-terminal and C-terminal lobes are held together by a middle module (MM) 3 (aa 729–774), a sensor of pH and [Ca²⁺] changes. The calcium binding module 3 is intrinsically disordered at low [Ca²⁺]. Upon Ca²⁺ binding, both the monomer (B) and dimer (BSP) become more compact. Millimolar [Ca²⁺] was shown to counteract the effects of varying pH on BSP and its subconstructs, and to antagonize pH dependent aggregation. This response of BSP to Ca²⁺ may be responsible to inhibiting S. aureus biofilm development by 10–12 mM Ca²⁺ [37], a concentration consistent with the Ca²⁺ concentration in milk [38]. Since the levels of free Ca²⁺ fluctuate in different body liquids, for example, it is maintained about 1.1 and 1.3 mM in mammalian blood [39], Bap was suggested to act as a Ca²⁺ and pH sensor through disorder-to-order conformational switches that conferred the ability of S. aureus to sense environmental signals and adapt to environmental conditions[39].

In contrast to B. subtilis and Staphylococci sp., the functional amyloid, FapC [40], is a part of the biofilm matrix. It promotes biofilm formation in P. aeruginosa but hasn’t been implicated in calcium binding.

eDNA:

Extracellular genomic DNA (eDNA) is a crucial structural component in many bacterial biofilms that can productively interact with calcium cations [41]. DNase can inhibit biofilm formation or disrupt established biofilms of multiple microbial species including B. cereus [42, 43]. In S. aureus, Staphylococcus epidermidis, Vibrio cholera, and P. aeruginosa PAO1, the DNase-driven disruption of established biofilms was dependent on biofilm age; young biofilms were more sensitive to DNase than older biofilms [42, 44, 45]. Thus, eDNA is a vital component for biofilm structure and maturation. eDNA was shown to interact with other ECM components, such as the P. aeruginosa PEL exopolysaccharide and other protein components, adding to structure stability [41]. In a biofilm, eDNA is released from lysed cells or via regulated eDNA secretion mechanisms. For example, in S. aureus, cell lysis is controlled by the cidA-lrgA holin-antiholin system [46] The abundance of eDNA could affect calcium carbonate deposition in biofilms as in vitro, DNA molecules mediated the nucleation and growth of the inorganic phase of calcium carbonate[47].

Direct evidence for the contribution of extracellular DNA (eDNA) to calcium carbonate precipitation was recently obtained in B. cereus. This bacterium produces an organic extracellular matrix containing negatively charged eDNA that was suggested to act as a proficient nucleator for calcium carbonate mineralization [48, 49]. In this bacterium, the accumulation of extracellular DNA (eDNA) and the precipitation of calcium carbonate required the presence of both calcium and DNA in the medium. At the same time, other polymers were independent of CaCO3. Removal of eDNA by DNase did not affect other matrix components (exopolysaccharides and proteins) but resulted in a six-fold decrease in the abiotic precipitate yield. The formation of calcium carbonate was also accelerated after the addition of exogenous salmon sperm DNA. In many biofilms, the negatively charged eDNA lattice can promote local Ca²⁺ supersaturation, which, together with an increase in the concentration of carbonate ions, may promote the formation of an insoluble precipitate of calcium carbonate [49]. These results indicated that exopolysaccharides and eDNA independently impact calcium carbonate precipitation in biofilms.

Exopolysaccharides, surfactants, and poly-γ-glutamic acid:

P. aeruginosa strains can synthesize at least three different exopolysaccharides, Pel, Psl, and alginate, composing the biofilm matrix [22]. While the ability to do so varies between strains and clinical isolates [50], all polymers can interact directly or indirectly with calcium. It was shown that P. aeruginosa biofilms induce substantial in situ precipitation of calcite [51, 52] and that, similarly to B. subtilis, the deposits produced were morphologically distinct from abiotically precipitated calcium carbonate particles. Under supersaturated conditions, the in situ biomineralization occurred with abiotically formed calcium particles trapped in P. aeruginosa biofilms [51]. Both processes immobilize inorganic carbon and contribute to microbial carbonate sequestration. No carbonate biomineralization occurred in the biofilm matrix exopolysaccharide mutant strain PAO1 Δpel Δpsl, lacking two of the three exopolysaccharides, suggesting that these components of biofilm matrix contribute to carbonate precipitation[51]. Further, calcite biomineralization was not found in metabolically inert biofilms, expressing Pel and Psl [51]. These results highlight the possibility that in non-mucoid PAO1, active metabolism, rather than exopolymeric substances binding, is the predominant mechanism for heterogeneous carbonate biomineralization in biofilms [51, 52]. In support of this notion, some metabolic features that are enhanced in biofilm cells but not in single cells were associated with increased biomineralization. For example, the expression of Urease(UreA) was enhanced in biofilms. This induction was likely due to the enhanced amino acid catabolism in nitrogen-deprived cells, as the availability of nitrogen was affected by the altered diffusion within biofilms [53].

β-carbonic anhydrases were also shown to be differentially regulated in biofilm and planktonic cells in response to elevated levels of calcium and carbon dioxide ([25] (unpublished data)). It is interesting to determine whether the P. aeruginosa Pel and Psl exopolysaccharide matrix components affect respiratory conditions within the biofilm to induce the expression of carbonic anhydrases. Since the biofilm microenvironment maintains an oxygen gradient and, thereby, a potential carbon dioxide gradient [54], Pel and Psl may indirectly limit the diffusion of carbon dioxide from aerobic respiration. Considering that tightly packed P. aeruginosa biofilm aggregates producing a Psl and Pel rich biofilm matrix were found to be commonly present in CF sputum and lung samples [55, 56], understanding the potential link between the accumulation of Pel-Psl containing matrix and increased calcium carbonate deposition warrants investigation.

While the metabolism-matrix-calcium carbonate interactions in non-mucoid Pseudomonas strains remain to be resolved, mucoid P. aeruginosa strains may respond more directly to calcium. These strains overproduce the negatively charged polysaccharide alginate. The elevated levels of calcium that are commonly detected in CF sputum [57] were shown to increase the expression of alginate biosynthetic genes and lead to the accumulation of alginate in the matrix of significantly enhanced (10–20 fold) biofilms [58]. More recently, Jacobs et al. provided details on the molecular mechanism of calcium-alginate interactions by comparing three isogenic P. aeruginosa strains, PAO1 (nonmucoid; AlgU is inactivated), PAO1 mucA22 (mucoid; AlgU is active), and PAO1 mucA22 ∆algD (nonmucoid mutant where AlgU is active, but alginate cannot be produced) [59]. The mucoid strain PAO1 mucA22 produced gelled biofilm in the presence of calcium, but without calcium, formed comparatively small aggregates. These results suggested that alginate gelation occurs by calcium cross-linking between the negatively charged alginate polymers. Consistently, calcium-gelled mucoid biofilms were frequently thicker than their non-gelled counterparts. This, together with the elevated levels of calcium in the CF sputum, supports the hypothesis that calcium regulation of alginate biosynthesis and its interactions with produced alginate occur in vivo in patients.

Additional regulators of calcium-exopolysaccharides interactions are microbial surfactants. The role of P. aeruginosa - produced surfactants rhamnolipids in calcium carbonate precipitation was exposed during the investigation of the interactions between alginate and calcium carbonate in a synthetic system (i.e., with no bacterial cells) [60] . Importantly, the effect of these biosurfactants was dependent on their concentration and whether they are present directly in the hydrogel matrix or the carbonate solution surrounding the hydrogel [60]. The highest increase in vaterite (calcium carbonate polymorph) content, as well as specific surface area and porosity (allows the passing through of liquid or vapor), were observed when rhamnolipid micelles were present directly in the CaCl2 cross-linked hydrogel at a concentration of 0.05 M. The mechanism of CaCO3 precipitation in pure alginate hydrogel containing rhamnolipids has been proposed to rely on cross-linking, resulting in a gelled biofilm matrix [60]. The production of rhamnolipids is conditional, and their concentrations vary in different stages of biofilm formation [61]. However, it remains to be determined how local and temporal rhamnolipids production is regulated and how it affects calcium carbonate deposition and alginate gelation in P. aeruginosa biofilms.

In B. subtilis, the shape and molecular organization of extracellularly precipitated calcium carbonate are strongly hampered by deletions in the tyrosine kinase PtkA (previously ywqC) that regulates exopolysaccharide biosynthesis [13]. In addition, B. subtilis exopolymers often contain negatively charged molecules that bind calcium. For example, B. subtilis exopolysaccharides are enriched with negatively charged uronic acids, which have a high affinity for calcium [62]. Another example is poly-γ-glutamic acid (γ-PGA) [16]), a water-soluble, edible, nylon-like Bacillus-derived exopolymer strongly associates with calcium ions [63]. B. subtilis biofilms are also natural producers of a potent surfactant, designated surfactin [64], whose role in calcium-mediated gelation of exopolysaccharides remains to be determined. Interestingly, an interaction between calcium and surfactin was already suggested to play a role in biofilm development [65].

Although calcium was shown to interact with the extracellular polymers produced by diverse bacteria, and the accumulation of calcium carbonate was dependent on the organic extracellular matrix, more research is needed to fully characterize the regulation and the complex various, multifaceted interactions between the organic and inorganic components of matrixes in microbial biofilms.

Calcium Signaling Plays a Role in Shaping the Transcriptome of Bacterial Biofilms

Among the multiple roles of calcium in biofilms, its role as a signal is the least studied. In eukaryotes, calcium signaling is a key mechanism for intracellular communication and controls a wide variety of essential cellular processes ([66, 67]). This mechanism relies on the tightly regulated homeostasis of intracellular Ca²⁺ concentration (termed [Ca²⁺_in]). In bacteria, Ca²⁺ has also been shown to control several vital physiological processes, including those relevant to biofilm development. Among these processes, chemotaxis, cell differentiation, membrane transport (channels, primary and secondary transporters- see below), virulence, and host-pathogen interactions were documented [68].

We and others established key mechanistic prerequisites for Ca²⁺_in signaling in bacteria exemplified by P. aeruginosa. Similarly to eukaryotes, P. aeruginosa tightly controls the [Ca²⁺_in] at low micromolar level and, in response to the elevated extracellular calcium concentration (termed [Ca²⁺_ex]), generates a transient increase in [Ca²⁺_in] that returns to the resting state within minutes, even when the [Ca²⁺_ex] is three orders of magnitude higher than [Ca²⁺_in] [69]. Several key transporters required for the maintenance of Ca²⁺_in homeostasis have been identified in P. aeruginosa [69]. Recently, a Ca²⁺ channel, CalC, that is required for Ca²⁺ regulation of gene expression in P. aeruginosa was identified (Guragain et al., submitted). CalC mutant failed in forming complex 3D structures of biofilm colonies [21]. Supporting this observation is the increasing evidence of the prevalence of bacterial calcium-binding proteins [69] that may be involved in intracellular Ca²⁺ signaling. Proteomic and transcriptomic studies in P. aeruginosa and B. subtilis showed that changes in Ca²⁺ availability alter the expression of hundreds of genes [14, 70]. Still, the mechanisms are much better resolved. P. aeruginosa. These results support the premise that a functional Ca²⁺_in signaling system exists in bacteria and set the stage for the next goal of determining the molecular details of Ca²⁺ sensing and signaling that controls cellular responses.

Bacteria are also able to recognize and transmit extracellular Ca²⁺ signal. Several types of sensing systems are involved in these processes. The first type includes bacterial two-component regulatory systems commonly consisting of a sensor kinase and a transcriptional activator. In P. aeruginosa, PhoQ is a Ca²⁺ and Mg²⁺ sensor that modulates transcription in response to these cations. In addition to Ca²⁺ and Mg²⁺, PhoQ can bind Mn²⁺. This binding keeps the protein in a repressed state, where it inhibited the transcription of many virulent genes [71]. Another two-component regulatory system, CarSR is positively regulated by elevated Ca²⁺ and controls Ca²⁺ dependent regulon including a putative nucleotide-binding carP [70] that is required for maintaining the basal level of Ca²⁺_in and Ca²⁺-dependent induction of pyocyanin production, tobramycin and oxidative stress resistance, as well as swarming motility [72]. CarSR from PAO1 is identical to its homolog BqsSR from PA14, and both responded to iron [73].

The second type of Ca²⁺ sensor is LadS, a hybrid membrane-bound sensor, containing a histidine kinase domain and a periplasmic Ca²⁺-binding DISMED2 domain [74]. In response to Ca²⁺, this protein stimulates the Gac/Rsm pathway and triggers the switch from acute-to-chronic behavior of the pathogen [74].

Finally, the EF-hand domain-containing Ca²⁺ sensor EfhP that possesses several key features of a eukaryotic Ca²⁺ sensor calmodulin was discovered to undergo Ca²⁺ dependent structural rearrangements and contribute to the Ca²⁺-dependent transcriptional regulation of a large number of P. aeruginosa genes [75] [76]. Interestingly, transcription of efhP is regulated by Ca²⁺ and iron, which sets the sensor at a regulatory crossroad between calcium and iron signaling. The dual response of both CarSR and EfhP to calcium and iron suggested an intimate connection between the two metals in the coevolution of P. aeruginosa adaptation. These findings strongly suggest that extracellular calcium serves as a signal. The mechanisms for this signaling pathway and their dependence on membrane-associated receptors remain to be further explored.

In B. subtilis, calcium also acted as a signal and induced biofilm formation and the production of extracellular matrix [13, 14]. However, the exact mechanism remained unknown. Calcium signaling was suggested to involve three calcium transporters: YloB (ATP-driven Ca²⁺ pump) [77], ChaA (H+/ Ca²⁺ exchanger) [78], and YetJ (pH-dependent calcium leak channel) [79]. Mutating YloB alone impairs biofilm formation and calcium deposition, however, a dual mutant for ChaA and YetJ remains to be examined. The regulatory role of Ca²⁺_ex was manifested by the transcriptome response: about 20% of the genome (n=875) was shown to be induced in biofilm cells in response to Ca²⁺_ex. Calcium-induced genes included those associated with biofilm formation and extracellular matrix biosynthesis [13]. A follow-up transcriptome study determined that calcium induces the tapA-sipW-tasA operon, exopolysaccharide biosynthesis, and sporulation [14]. In contrast, genes associated with motility were repressed [14]. The response of the Ca²⁺_in concentration to the addition of Ca²⁺_ex ([80] supported the hypothesis that Ca²⁺ regulation in B. subtilis also involved Ca²⁺_in signaling, but the involved signaling cascades and mechanisms remained to be determined.

A transcriptome study revealed that Ca²⁺_in homeostasis plays a crucial role in regulating genes involved in the synthesis of lignocellulolytic enzymes and exoenzymes, especially under graphene oxide stress [80] . The use of Ca²⁺ chelator (EDTA) and deletion of spcF (encoding a calmodulin-like protein essential for stage III sporulation) eliminated the effect of Ca²⁺_ex on gene expression [80]. So far, no phenotype for calmodulin-like proteins in biofilm development has been reported in B. subtilis, although genes regulated downstream to spcF were suggested to play a role in biofilm formation. For example, exoproteases (regulated by SpcF) can support extracellular matrix remodeling and nutrient supply [81].

While the molecular mechanisms of calcium homeostasis in B. subtilis are mainly unresolved, Keren-Paz et al., found that the formation of calcium-dependent biofilms was impaired in ΔyhdQ (previously CueR), a mutant of poorly characterized transcriptional regulator belonging to the metal-responsive MerR family[82]. This mutant had a distinct defect in biofilm development and regulation of all known calcium transporters [82]. Transcriptome analysis revealed that YhdQ null mutant affected pathways involved in both magnesium [14, 83] and calcium homeostasis [14]. Although the specificity of the activation signal for YhdQ remained unclarified, the alteration of the calcium-dependent transcriptome in the absence of YhdQ [14] suggested that calcium may serve as at least one of them.

Intracellular Calcium Storage and its Role in Calcium Deposition

Intracellular calcium carbonate accumulation (most likely as amorphous calcium carbonate ACC rather than as a crystalline material) was described in the Gram-positive bacteria B. licheniformis [48] and B. subtilis, where it involved the calcium transporter YloB and the response regulator YhdQ [14]. In B. subtilis, intracellular calcium storage was detected in cells from structured biofilms formed under calcium-rich conditions [14]. It was shown that the frequency of cells with noticeable intracellular calcium storage increased within the structured colony areas [14]. Intracellular calcium carbonate storage was also documented in photosynthetic and magnetotactic bacteria [9, 10, 84]. The local storage could be due to the differential availability of calcium, carbonate, a potential local nucleator, or micro-alkaline pH within the cell [8]. All these possible causes for ACC accumulation within the cell are not mutually exclusive.

Intracellular calcium storage is expected to affect extracellular matrix calcification directly or indirectly. For example, lysed cells may release ACC to interact with the organic extracellular matrix. Alternatively, ACC can be actively secreted to promote matrix mineralization [14]. Internal ACC storage may also affect intracellular calcium signaling, change global and local cytoplasmic calcium concentrations, and play a significant role in the overall calcium-cells relationship.

Harvesting Calcium Signaling and Calcium Deposition for Sustainable Construction and the Engineering of Probiotics

Resolving the molecular mechanisms by which B. subtilis interacts with calcium and vice versa is highly relevant to environmental biotechnology, as microbial biomineralization can be utilized to produce environmentally friendly biocement, enhancing its durability and rigidity [11]. Bacterial producers of cementous materials can also be used to restore heavily damaged calcareous stones from monuments and artworks [85]. This application is of great environmental importance as the production of cement (composed primarily of calcium carbonate) is responsible for about 8% of the world’s carbon dioxide emissions [86]. In sharp contrast, carbon dioxide is sequestered during biomineralization. Thereof, carbonate-mineralizing cells and their EPS (to achieve the desired cement properties) can reduce carbon dioxide emissions from the construction industry.

Crystals formed over the organic templates that can enhance biocement properties are either vaterite (generated by lactobacillus and cyanobacteria), aragonite (by corals and cyanobacteria) [87] or calcite (by B. subtilis and related species) [11]. These biomineralization systems offer a clear advantage to the construction industries by providing building blocks for self-healing cementitious materials [88]. Furthermore, Bacillus related species are easily genetically manipulatable. Therefore, the increasingly resolved genetics of calcium carbonate opens numerous pathways to utilize synthetic biology for carbon-neutral or low-carbon construction. In addition, B. subtilis biofilms are potent biocontrol agents, serving to repel viral, microbial, and fungal pathogens from crops (as reviewed extensively in [15, 16]). Thereby, the discovery of calcium-dependent biofilm formation in the Bacillus clade (including phylogenetically related Bacillus species with similar properties) [89] is expected to contribute to sustainable agriculture.

In addition to sustainable construction and agriculture, mineralization was recently suggested to play a crucial role in the engineering of probiotics. Supplementation with beneficial microbes has been commonly applied as an appealing strategy to prevent or reduce pathogen colonization and to maintain a healthy microbial flora [90]. The effectiveness of microbial biotherapeutics could be hampered by a significant reduction of the viability of probiotics following their oral delivery to the intestine [91]. Therefore, protective methods are urgently needed for preparing oral living biotherapeutics. Geng et al. recently introduced a mineralization-dependent strategy to protect probiotic bacteria from external insults to tackle these challenges. Microbial bio-interface mineralization was used to deposit an ultra-resistant and self-removable coating for bacterial protection. The bacteria were coated by productive interactions between inorganic-organic hybrids mimicking biomineralization [92]. Single-cell coating with calcium minerals allowed the probiotic agents (Bacteroides fragilis) to resist insults associated with manufacture and product sterilization, including oxygen exposure, UV irradiation, and ethanol. After oral ingestion, gastric acid could rapidly neutralize low intragastric pH and promote the demineralization-triggered release of coated bacteria. These findings were extended to other probiotic strains including Escherichia coli Nissle 1917, the obligate anaerobe Bifidobacterium, and Lactobacillus rhamnosus GG. This work suggested that bio-interface mineralization but also biomineralization can open a window for preparing multifunctional bacterial-based bioagents for various biomedical applications [92]. In this context, it is interesting to consider the potential of the proficient calcite former B. subtilis as a biotherapeutic and probiotic agent [93, 94].

Clinically relevant applications of Calcium Signaling and Calcium Deposition

Calcium deposition was shown as a potential factor altering the resistance of biofilms to antibiotics [8] and the 3D structure of biofilms, though other minerals may serve similar functions. These outcomes have a major impact on the clinical aspects of bacterial biofilms. For example, deposition of calcium phosphate crystals within Proteus mirabilis, P. vulgaris, and Providencia rettgeri biofilms blocks catheters in infected patients [95–97], and calcium phosphate mineralization (mediated by Urease) was shown to increase P. mirabilis resistance to ciprofloxacin [98] and increase its survival in dual-species biofilms [99]. The physiological role of calcium phosphate deposits within these medical biofilms and whether dedicated cells facilitate this process remains to be determined. While calcium deposition’s role in the virulence of Gram-positive and Gram-negative pathogens remains to be further studied, calcium signaling in P. aeruginosa is robustly linked to biofilm formation and virulence. Several Ca²⁺ regulatory and signaling network components may serve as potential drug targets. They include calcium sensors (e.g., EfhP [70, 72] and LadS [74]), calcium transporters (e.g., CalC)[21], calcium-responsive two-component systems (e.g., CarSR)[70] and their regulatory targets with pleotropic impacts on P. aeruginosa virulence, such as CarP [70]. However, further studies are imperative to validate these candidates for specific drug development.

Concluding Remarks

The evidence emerges for the role of extracellular calcium and intracellular calcium signaling in biofilm formation in environmentally and clinically important bacteria, including two examples of strong biofilm formers, B. subtilis and P. aeruginosa (Figure 1). In the former, calcium regulates the gene expression of motility genes and extracellular matrix genes. In the latter, calcium signaling controls genes associated with host colonization and virulence. Connections between calcium and iron/redox sensing were observed for both microorganisms. It is feasible that this coupling reflects a frequent co-occurring of the two stressors, for example, the high abundance of calcium cations in the CF sputum and the increased levels of iron commonly generated by neutrophils and macrophages during the respiratory burst to combat pathogens.

Figure 1. Extracellular and intracellular roles of calcium in microbial biofilms.

Calcium influx through calcium channels at the cellular membrane connects extracellular and intracellular calcium in P. aeruginosa and B. subtilis. In addition, calcium negatively regulates by direct interactions with the adhesin Bsp of Staphylococci but can also induce the transcription of the TasA adhesin of B. subtilis. Calcium and calcium carbonate interact with exopolysaccharides in the extracellular environment. Calcium ions interact with alginate, eDNA, and matrix fibrils. Calcium may act to cross-link exopolysaccharides but is also affected by all negatively charged components of the extracellular matrix, providing additional nucleation sites for biomineralization. Surfactants may have essential roles in determining the porosity of the formed minerals. Within the cells, calcium homeostasis and signaling dictate the transfer of cells from free-living (planktonic) cells to adherent/matrix-producing/virulent cells. Calmodulin-like proteins mediate some but not all calcium-dependent gene expression (Calmodulin-like protein image was imported from hypermol). The figure was generated by Biorender software. Calmodulin image was imported from PDB-101 [100], and calcium carbonate images from [13].

Calcium signaling may also provide adaptive regulatory wiring to protect against oxidative stress, as shown in the soil bacterium B. subtilis. The continuous investigation of these signaling systems may highlight the conservation of calcium signaling principles in all domains of life and discover conserved protein functions that mediate calcium-dependent circuits. While considering the importance of calcium and calcium homeostasis as regulators of gene expression, the role of calcium deposition in morphogenesis must be regarded as equally important (Figure 1). It is an exciting and vital challenge to continue developing these complementary research directions. Since these pathways can be utilized in various applications ranging from biotechnology and ecology to construction and medicine, their importance cannot be overestimated.

Outstanding Questions.

• What are the roles of calcium in biofilm development?

• What are the specific contributions of calcium signaling and calcium-matrix interactions to biofilm development?

• Which extracellular matrix components are essential for biomineralization in biofilms, and how are these interactions spatio-temporally regulated?

• What are the exact pathways by which calcium signaling regulates gene expression in bacteria?

• How does intracellular calcium mineralization affect calcium homeostasis?

• How can we utilize microbial mineralization to generate more efficient biocementous materials?

• How can calcium sensory systems be targeted to control acute biofilm infections by Pseudomonas aeruginosa?

Highlights.

• Calcium signaling is an essential mediator of gene expression in all-developmental systems.

• Intracellular and extracellular calcium recently emerged as regulators of biofilm formation in bacteria and were characterized in more detail for Pseudomonas aeruginosa and Bacillus subtilis.

• Calmodulin-like proteins play a fundamental role in calcium-dependent gene expression in Pseudomonas aeruginosa and Bacillus subtilis.

• The formation of calcium carbonate extracellular structures is associated with complex colony morphology and altered diffusion in microbial biofilms.

• A broad range of extracellular matrix polymers, including functional amyloids, eDNA, and exopolysaccharides, interact with calcium and promote calcium carbonate mineralization.

• The genetic pathways underlying microbial mineralization open pathways to utilize synthetic biology for carbon-neutral or low-carbon biocement for construction.

• Several Ca²⁺ regulatory and signaling network components may serve as potential drug targets, including calcium channels and calcium-responsive two-component systems.