**2. Ca2+ homeostasis in bacteria**

levels of free cytosolic calcium are regulated by Ca2+-binding proteins, primary and secondary

Although the role of Ca2+ in prokaryotes is still unclear, there is increased evidence favoring

several bacterial physiological processes including: chemotaxis, cell differentiation such as spore development and heterocyst formation, membrane transport (channels, primary and secondary transporters), virulence and host pathogen interactions [4, 6–10]. Similar to eukaryotes, bacteria maintain cytosolic free Ca2+ within the nM range even in the presence of mM extracellular Ca2+ [11–15]. Ca2+-stimulus-response has been documented during environmental stress, toxicants [16–18] carbohydrate metabolites [19, 20], iron acquisition, quinolone signaling and type III secretion, which are secretory systems comprised of proteins found in pathogenic Gram negative bacteria that are used to infect eukaryotic cells [21, 22]), suggesting that Ca2+ signals are relevant to microbial physiology. Primary and secondary transporters

show that the level of similarity with eukaryotic counterparts is striking. For example sodium channels show high degree of conservation but their structure is simpler [23]. The ATPase found in *B. subtilis* is analogous to the typical eukaryotic type IIA family of P-type ion-motive ATPases [24]. However, direct evidence that these transporters regulate the concentration of cytosolic free Ca2+ is limited. There is evidence of calcium binding proteins (CaBP) in several genera of bacteria, including proteins with EF-hand domains [25, 26], and other calcium motifs such as β-rolls motif, Greek key motif, repeats in toxin and Big Ca2+ domain [27–30] but their functional role needs to be investigated. Proteomic and transcriptomic studies in *E. coli*,

) in signal transduction in bacteria. Indirect evidence shows that Ca2+ affects

) have been identified in various genera of bacteria. Data

transporters and cytosolic Ca2+ stores preventing calcium phosphate toxicity [1, 3].

a role for ([Ca2+]

90 Calcium and Signal Transduction

i

including channels (Ca2+, K+

**Figure 1.** Possible roles of calcium in bacteria.

, Na+

Initial measurements of [Ca2+]i in bacteria were a challenge because of the unique physical characteristics of bacterial cells (tiny size, cell walls and membrane), the difficulty in manipulating live cells and the toxicity of reagents [13, 33]. Other concerns included those associated with Ca2+ research such as contamination and lack of selectivity of Ca2+ chelators [34–36]. With the introduction of molecular technology, the photoprotein aequorin gene was expressed in bacterial cells to measure cytosolic free Ca2+ in live cells. In this way, several investigators were able to continuously monitor cytosolic free-Ca2+ in several genera of bacteria [12–14]. A crucial discovery was that all bacteria tested maintained very low levels of cytosolic free Ca2+, even in the presence of 1–10 mM extracellular Ca2+ (**Figure 2**). Cytosolic free Ca2+ in bacterial cells ranges from 100 to 300 nM, very similar values to those observed in eukaryotic cells [11, 13, 14]. These findings suggest that microbial cells must have transport systems (influx and efflux), proteins or other structures that may serve as intracellular free Ca2+ targets that may play a role in the maintenance of Ca2+ homeostasis.

The role of channels, ATPases and exchangers in Ca**2+** homeostasis has not been investigated critically and none of these have been experimentally proven to transport Ca2+ specifically. The contribution of bacterial CaBP to Ca2+ homeostasis remains undetermined [26, 37]. However, recent work shows that the disruption of particular ATPases (PA2435, PA3920), the exchanger (PA2092) and a putative EF-hand protein, is evidence that these transporters are necessary to maintain low intracellular Ca2+ levels in *P. aeruginosa* [15, 38]. A proteomic analysis in *B. subtilis* showed that several cytosolic proteins appear to bind Ca2+, as determined by Ca2+ autoradiography [32]. Some of these proteins, identified by liquid chromatography/ mass spectrometry include: a potential cation transport ATPase, fructose biposhate aldolase, DnaK 70 and adenylate kinase. These proteins were induced when cells were treated with extracellular divalent cation chelator ethylene glycol tetraacetic acid (EGTA) and reduced when treated with high extracellular Ca2+. None of these proteins however had Ca2+ binding domains [32]. Notably genes encoding fructose biposhate aldolase, DnaK 70 and adenylate

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

and K+

) and anion (Cl<sup>−</sup>

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 *Thermoplasma volcanium* are activated at millimolar Ca2+ concentrations questioning the physiological relevance since Ca2+ signals occur within micromolar range. On the other hand,

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

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

ers. 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

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].

antiporters [59] and Ca2+/PO4

In most bacteria, Ca2+ is apparently exported by Ca2+ exchangers, Ca2+/H+

[57, 58], Ca2+/Na+

channels of the archaea *Methanobacterium thermoautotrophicum*

) channels, ATPases and exchangers

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

Calcium Signaling in Prokaryotes

93

channel in cyanobacteria resulted in increased

or Ca2+/Na+ antiport-

3+ symporter respectively.

**3.1. Influx**

The existence of cation (Na+

and the Ca2+-dependent K+

deletion of the SynCaK, a Ca2+-dependent K+

putative channel is shown in **Figure 3**.

Ca2+ fluxes, similar to eukaryotic cells.

**3.2. Efflux**

as potential Ca2+/H+

**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 different CaCl<sup>2</sup> concentrations: 0.5, 1, 5, and 15 mM. J Anal Bioanal Tech reproduced with permission.

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 buffering intracellular free Ca2+.

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 spectroscopy.

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+.
