**5. Ca2+ signaling**

The hypothesis that Ca2+ acts as a messenger in bacterial cells is based on the observation that environmental signals induce changes in the level of cytosolic free Ca2+. Microorganisms must quickly adapt to changes in the environment in order to survive. Therefore, bacteria must have evolved sophisticated regulatory networks to constantly monitor signals that are critical for their continued existence. How bacterial cells sense the external signal has not been determined yet but experimental observations suggest that may occur through different mechanisms including: cytosolic-free Ca2+ transients, membrane sensors, two component systems and its regulatory proteins, and Ca2+ sensors transducing the signal.

Over the years, evidence of a Ca2+-mediated stimulus response in bacteria has been documented. Since 1977, Ordal reported that cytosolic Ca2+ controlled the rotation of the flagella in *B. subtilis cells*. Later work corroborated that cytosolic Ca2+ transients affect bacterial motility in *E. coli*, possibly through the phosphorylation of the Che proteins [78–80]. The involvement of Ca2+ as a signal transducer in a variety of environmental conditions, where cytosolic free Ca2+ is elevated as a result of the stimulus, has been shown in various organisms including: oxidative stress in *B. subtilis* [81], heat/cold shock, and salt and osmotic stress in *Anabaena* strain PCC7120 [14, 82], carbohydrate fermentation products in *E. coli* [19], organic solvents, pharmaceuticals and antibiotics in cyanobacteria [16, 17].

Evidence that membrane-bound proteins may be able to transduce Ca2+ signal was shown *in vitro* using the chimeric protein Taz1. Under low concentrations of Ca2+, Taz was phosphorylated leading to the activation of porin genes in *E. coli* [83, 84]. No *in vivo* studies have been followed up. A more recent report in *Vibrio cholera*, showed that Ca2+ greatly enhances the transmembrane virulence regulator (TcpP) activity by increasing protein-protein interaction in the presence of bile salts, leading to the activation of downstream virulence factors [10].

Two component regulatory systems, consisting of a sensor kinase and a transcriptional activator, are commonly used by bacteria to sense and respond to environmental signals. Several of these systems have been shown to respond to extracellular Ca2+. In the PhoPQ system in *Salmonella typhimurium* and *P. aeruginosa*, PhoQ is a Mg2+, Ca2+ sensor that modulates transcription in response to cation levels. The binding of PhoQ to Ca2+, Mg2+ or Mn2+ keeps the protein in a repressed state inhibiting the transcription of many virulent genes [85, 86]. In *V. cholera*, the *ca*lcium *r*egulated *s*ensor (carS) and regulator (carR) were shown to be decreased when bacterial cells grew in Ca2+ supplemented medium. Further analysis demonstrated that expression of vps (*Vibrio* polysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system [87]. In *V. parahemolyticus*, Ca2+ influences gene expression for type III secretion systems (T3SS<sup>1</sup> ) and swarming. A transcription factor called CalR was shown to repress T3SS1 and swarming, which in turn were linked to a σ54-dependent regulator [22]. Another two-component system AtoS-AtoC, which mediates the regulation of PHB complexes in *E. coli* is induced by Ca2+. It was shown that the highest accumulation of PHB complexes occurred in AtoS-AtoC expressing *E. coli* cells compared to deletion mutants AtoSC at high Ca2+ concentration in cytosolic and membrane fractions [88, 89]. More recently, in *P. aeruginosa*, the two-component regulator PA2656-PA2657 genes were induced by CaCl2 . Deletion mutations and transcriptome analysis revealed that this two-component system may be responsible for regulating the expression of periplasmic proteins and affecting Ca2+ homeostasis [90].

**5. Ca2+ signaling**

**Organism Protein name Accession** 

*Thermotoga maritime* 4-α-Glucano-

96 Calcium and Signal Transduction

*Bacillus anthracis* Protective

*Salmonella typhimurium* Periplasmic

*Sphingomonas* sp Periplasmic

**number**

*Saccharopolyspora erythrea* Calerythrin P06495 177 Helix-loop-

*Rhizobium etli* Calsymin Q9F6V9 293 Helix-loop-

*Escherichia coli* B Slt35 P41052 361 Helix-loop-

*Clostridium thermocellum* Dockerin A3DCJ4 350 Helix-loop-

galactose binding

alginate binding protein

*Pseudomonas aeruginosa* Alkaline protease Q03023 479 Strand-

*Halothermothrix* α-Amylase A Q8GPL8 515 Strand-

transferase

antigen

protein

**a.a. number**

P80099 441 Helix-loop-

P13423 764 Helix-loop-

P23905 332 Helix-loop-

Q9KWT6 526 Helix-loop-

**EF-hand/ EF-handlike motif**

helix

helix

helix

helix

helix

helix

strand

loop

loop-strand

loop-helix

**Potential role of Ca2+**

Buffer [4, 24]

Transducer [4, 36]

Unknown [4, 24]

Structural [4, 24]

Structural [4, 23]

Structural [4, 23]

Structural [4, 23]

Regulatory [4, 23]

Unknown [4, 24]

Structural [4]

**Refs.**

transducing the signal.

The hypothesis that Ca2+ acts as a messenger in bacterial cells is based on the observation that environmental signals induce changes in the level of cytosolic free Ca2+. Microorganisms must quickly adapt to changes in the environment in order to survive. Therefore, bacteria must have evolved sophisticated regulatory networks to constantly monitor signals that are critical for their continued existence. How bacterial cells sense the external signal has not been determined yet but experimental observations suggest that may occur through different mechanisms including: cytosolic-free Ca2+ transients, membrane sensors, two component systems and its regulatory proteins, and Ca2+ sensors

Protein accession numbers in UniProtKB database. Reproduced with permission from Elsevier. Dominguez et al. [4].

**Table 1.** Examples of bacterial proteins containing EF-hand and EF-hand-like motifs with known structure.

Over the years, evidence of a Ca2+-mediated stimulus response in bacteria has been documented. Since 1977, Ordal reported that cytosolic Ca2+ controlled the rotation of the flagella in Bacterial CaBP that may be involved in in signal transduction include CabC, which may be regulating spore germination and aerial hyphae formation in *Streptomyces coelicolor* [91]. The recently reported EfhP from *P. aeruginosa* that is required for Ca2+ homeostasis [38] and other two EF-hand proteins from *S. coelicolor* and *S. ambofaciens* whose function remains to be discovered [92, 93].

Despite all the information accumulated over the past few years, Ca2+ signaling in bacterial physiology remains to be elucidated. Further work is needed to uncover the specific nature of the Ca2+ signal transduction, its components and their specific regulation and function.
