**4. Bacterial Ca2+ binding proteins (CaBP)**

P and F-type Ca2+ ATPases have been described in bacteria. ATPases that were purified and

**Figure 3.** Coordination geometry of Ca2+ in the PHB-PP helix. (A) Calcium forms ionic bonds with four phosphoryl oxygens of poly-P and ion-dipole bonds with four ester carbonyl oxygens of poly-hydroxybutyrate (PHB) to form a neutral complex with distorted cubic geometry. (B) Computer model horizontal cross section showing the poly-P helix with the poly (HE) helix with Ca2+ surrounded by the oxygen moieties of both polymers. The seven Ca2+ displayed are from two turns of the poly-P helix. Light blue, hydrogen; dark blue, carbon; red, oxygen; green, phosphorous; aqua, Ca2+. (C) View down the poly(HB) cylinder. Ca2+ (closed circles) bound to carbonyl ester oxygens (open circles) in a pattern that links each turn of the helix alternatively to the proximal turns above and below. Reusch and Sadoff [45]. Courtesy of Reusch RN.

the P-type ATPase from *Synechocystis sp.* showed vanadate sensitivity, which appears to be homologous to eukaryotic SERCA [61, 62], the F-type ATPase from *Flavobacterium odoratum* also vanadate-sensitive, phosphorylated only in the presence of Ca2+ [63] and the *Listeria monocytogenes* ATPase, which has low Ca2+ affinity, and it is induced at alkaline pH [64]. The *in vivo* function of these proteins remains to be characterized. Other ATPases that have been identified by bioinformatics include: CaxP from *Streptococcus pneumoniae* [65], YloB from *B. subtilis* [24], PacL from *Synechococcus* sp. [66] and PA2435 and PA3920 from *P. aeruginosa* [15]. Work by Naseem et al. [20] demonstrated that ATP is essential for Ca2+ efflux, and there is a possibility that ATP may regulate Ca2+ efflux through an ATPase. It was shown that the gene atpD, which encodes a component of an F-type ATPase is required for a normal Ca2+ efflux function. Although no specific transporter was shown here, the result is important, indicating

Bacterial transporters have not been studied systematically and knowledge about these proteins is limited. It appears that prokaryotes have multiple transporters with some redundancy.

shown to translocate or have Ca2+-dependent phosphorylation include:

94 Calcium and Signal Transduction

that ATP is surely necessary for transport of Ca2+ by a still unknown ATPase.

If a change in cytosolic free Ca2+ is to have any effect on bacterial physiology, bacterial cells must have intracellular Ca2+ targets in addition to influx and efflux mechanisms. Identification of such intracellular Ca2+ targets remains elusive. Nevertheless, a number of prokaryotic CaBP have been discovered by a combination of approaches: molecular technology and bioinformatics. According to Zhou et al. [26], sequence analyses of prokaryotic genomes showed the presence of 397 putative EF-hand proteins. However, most of these proteins with a few exceptions (Calerythrin from *Saccharopolyspora erythrea*, Calsymin from *Rhizobium etli*, the *Brucella abortus* Asp24, *Streptomyces coelicolor* CabA, CabD and Ccbp from *Anabaena* sp.) are hypothetical proteins [37, 67, 68]. Few proteins have been studied biochemically and none of these have been characterized functionally.

Five classes of EF-hand motifs have been reported in bacteria. The typical helix-loop helix EF-hand structure seen in Calerythrin and Calsymin, the *ex*tracellular *Ca*2+-*b*inding *r*egion (Excalibur), which has a shorter loop containing 10 residue motif DxDxDGxxCE found in various bacteria, the longer 15 residue Ca2+-binding loop seen in the *E. coli* lytic transglycosylase B, and the fourth and fifth classes lacking the first or second helix as described in the *C. thermocellum* dockerin and the *Sphingomona* ssp. alginate-binding protein, respectively [25, 26]. **Table 1** presents the five classes of bacterial EF-hand and EF-hand-like motifs proteins with known structures. The presence of the Ca2+ binding motifs must be tested for functional necessity or for viability of the organism.

Other Ca2+ motifs found in various bacteria include the Ca**2+**-binding β-roll motif, which includes proteins containing a region referred as repeats-in-toxin (RTX) [27, 69, 70] and a family of proteins with a signature sequence Proline P-Glutamate E Polymorphic GC-rich Repetitive Sequence (PE\_PGRS) [71, 72], the Greek key motif present in the βγ-crystallin superfamily containing Ca2+-binding proteins in Eubacteria and Archaea [29, 73–76] and finally the Big domain motif comprising proteins with an immunoglobulin-like domains [30, 77]. Most of these proteins however, are extracellular proteins and some require Ca**2+** within the μM to mM range to bind compared to eukaryotic cells that have high Ca2+ binding affinity within lower μM to nM range. Nevertheless, reports have shown that cytosolic free Ca2+ in *E. coli* can increase to tens of micromolar without any loss of viability, suggesting that bacterial Ca2+ targets may have lower affinity for Ca2+.

Prokaryotic CaBP encompass a diverse group of proteins that exhibit great structural variety. Binding of Ca2+ may provoke folding to a functional state or may lead to protein stabilization. Structural characteristics of these proteins suggest they may act as buffers, may play a structural role and/or may function as sensors/signal transducers. Much more research is needed to characterize biochemically and genetically bacterial Ca2+-binding proteins offering exciting possibilities and a challenge for the future.


*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,

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+

tion 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

two-component system may be responsible for regulating the expression of periplasmic pro-

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 dis-

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.

. Deletion mutations and transcriptome analysis revealed that this

) and swarming. A transcrip-

Calcium Signaling in Prokaryotes

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http://dx.doi.org/10.5772/intechopen.78546

pharmaceuticals and antibiotics in cyanobacteria [16, 17].

influences gene expression for type III secretion systems (T3SS<sup>1</sup>

were induced by CaCl2

covered [92, 93].

teins and affecting Ca2+ homeostasis [90].

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.
