**3. Influx and efflux transport systems in bacteria**

#### **3.1. Influx**

The existence of cation (Na+ and K+ ) and anion (Cl<sup>−</sup> ) channels, ATPases and exchangers have been documented in several genera of bacteria [4, 51]. Despite high resolution structure of some bacterial channels the physiological function reminds unknown [7]. Several bacteria have mechanosensitive ion channels that have large conductances (nanosiemens range) thus it would be expected to allow Ca2+ into cells. However, gene knockouts of major mechanosensitive channels in *E. coli* (MscL and MscS) still showed large Ca2+ influx [2, 52] and the Ca2+-dependent K+ channels of the archaea *Methanobacterium thermoautotrophicum* and *Thermoplasma volcanium* are activated at millimolar Ca2+ concentrations questioning the physiological relevance since Ca2+ signals occur within micromolar range. On the other hand, deletion of the SynCaK, a Ca2+-dependent K+ channel in cyanobacteria resulted in increased resistance to heavy metals suggesting a physiological role for Ca2+-mediated channels [53].

So far the best evidence of a Ca2+ influx channel in bacteria is the nonproteinaceous complex polyhydroxybutyrate-polyphosphate (PHB-PP). The channel is highly selective for Ca2+ at a physiological pH [54]. This preference has been attributed to a high density negative charge along the polyphosphate backbone. The complexes are abundant in stationary phase and correlate with high rise in cytosolic Ca2+. These complexes have many characteristics of protein Ca2+ channels: voltage-activated, conduct Ca2+, Sr2+ and Ba2+ and are blocked in a concentration-dependent manner by La3+, Co2+ and Cd2+ [44, 45, 55]. However, the genes encoding the synthesis of PHB complex remain to be properly identified and characterized. A figure of the putative channel is shown in **Figure 3**.

More recently, Bruni et al. [52] employing a sensor that simultaneously reports voltage and Ca2+ showed that Ca2+ influx is induced by voltage depolarization in *E. coli*. These exciting findings support the idea that bacteria may sense their environment through voltage-induced Ca2+ fluxes, similar to eukaryotic cells.

#### **3.2. Efflux**

kinase were found to be modulated by Ca2+ in *E. coli* [20]. These findings suggest that perhaps other proteins and anionic protein groups yet to be characterized may be involved in buffer-

**Figure 2.** Cytosolic free Ca2+ in Bacillus subtilis cells. *B. subtilis* Cells were transformed with a plasmid containing the gene for the photoprotein aequorin. Light emission was recorded in a luminometer after challenging the cells with

concentrations: 0.5, 1, 5, and 15 mM. J Anal Bioanal Tech reproduced with permission.

Bacterial cells lack organelles such as endoplasmic reticulum and mitochondria, which function as Ca2+ sinks in eukaryotes. However, some bacteria contain membrane-bound vesicles (acidocalcisomes) and polyphosphate granules that accumulate and store Ca2+ [39–42]. Other structures that bind Ca2+ in significant amounts are DNA and the complex poly-(R)-3 hydroxybutyrate (PHB)-polyphosphate (PP) [43–45]. Moreover, the periplasmic space, which is a region between the inner cytoplasmic membrane and the bacterial outer membrane and that has been found in both Gram negative and Gram positive bacteria [46–48], is another structure that has been reported that may play a role in storing and buffering Ca2+ [49]. Intracellular free Ca2+ measurements within the periplasmic space in live *E. coli* cells revealed that this structure can store 3–6-fold Ca2+ with respect to the external medium [49]. Chang and co-workers [50] also demonstrated high concentrations of Ca2+ associated with the cellular envelope in *E. coli* cells as determined by X-ray mapping and electron loss

Altogether, the aforementioned data suggest that bacterial cells may have different mechanisms to maintain cytosolic Ca2+ homeostasis. Further work should be performed to elucidate

how and why bacterial cells maintain low levels of intracellular free Ca2+.

ing intracellular free Ca2+.

92 Calcium and Signal Transduction

different CaCl<sup>2</sup>

spectroscopy.

In most bacteria, Ca2+ is apparently exported by Ca2+ exchangers, Ca2+/H+ or Ca2+/Na+ antiporters. These are low-affinity Ca2+ transport systems that use the energy stored in the electrochemical gradient of ions. Ca2+ exchangers differ in ion specificity and have been identified in a number of bacterial genera [11, 56]. In *E. coli*, the proteins ChaA, YrbG and PitB were reported as potential Ca2+/H+ [57, 58], Ca2+/Na+ antiporters [59] and Ca2+/PO4 3+ symporter respectively. Knockout of corresponding genes showed no effect on either Ca2+ influx or efflux [19, 20] raising questions about the role of these proteins. Potential redundancy is not ruled out. More recently, the multidrug transporter LmrP from *Bacillus lactis* has a predicted EF-hand motif with a Kd = 7.2 μM and two acidic residues (Asp-235 and Glu-327) binding Ca2+. LmrP was shown to selectively bind Ca2+ and Ba2+ and mediates selective Ca2+ efflux via electrogenic exchange [60]. A predicted transporter PA2092 from *P. aeruginosa* might be involved in Ca2+ efflux since intracellular Ca2+ accumulates after disruption of the corresponding mutant [15].

Besides protecting from toxic effects the question arises is Ca2+ transport in bacteria linked to

Calcium Signaling in Prokaryotes

95

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

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

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

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+

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

signaling? What is the contribution of these transport systems in Ca2+ homeostasis?

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

been characterized functionally.

for viability of the organism.

targets may have lower affinity for Ca2+.

possibilities and a challenge for the future.

**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.

P and F-type Ca2+ ATPases have been described in bacteria. ATPases that were purified and shown to translocate or have Ca2+-dependent phosphorylation include:

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

Bacterial transporters have not been studied systematically and knowledge about these proteins is limited. It appears that prokaryotes have multiple transporters with some redundancy. Besides protecting from toxic effects the question arises is Ca2+ transport in bacteria linked to signaling? What is the contribution of these transport systems in Ca2+ homeostasis?
