Together with flourine, bromine, iodine and astatine, chlorine belongs to the seventh group in the periodic table of elements, the "halogens". Chloride is one of the most abundant anions on earth and its content can make up to 75% in non-saline soil 1 or 90% in saline soils 2 of all inorganic anions. Huge amounts of chloride can also be found in marine habitats where its concentration can reach up to 550 mM (in combination with the corresponding cation Na⁺). This represents about 95% of the solvated anions. Due to this ubiquitous availability it is reasonable to assume that chloride is involved in biological processes.

Halophilic organisms are abundant in nature and thrive in habitats where molar concentrations of salts (mainly NaCl) are present and, consequently, very low water potentials exist. However, every living cell is dependent on the availability of liquid water and therefore has to counterbalance the external osmotic conditions. To serve this purpose, and despite the different osmotic demands, only two main strategies have evolved in nature.

A very abundant strategy to counteract the loss of water under hyperosmotic conditions is the accumulation of compatible solutes 3456. Compatible solutes are per definition small, organic molecules that are soluble in water to molar concentrations and which do not interfere with cell metabolism 7. Although a great spectrum of compatible solutes is used in the three domains of life, almost all can be assigned to two chemical classes: (i) the amino acids and their derivatives, such as glycine betaine, glutamine, glutamate, proline, ectoine or N-acetyl-β-lysine 891031112131415 and (ii) the sugars and polyols, such as trehalose, glycerol, di-myo-inositol-1,1'-phosphate (DIP) or sulfotrehalose 16171819202122. The accumulation can be accomplished either by uptake from the medium or by de novo synthesis.

Contrary to the accumulation of compatible solutes, some halophilic, anaerobic Bacteria (Halanaerobiales) and some Archaea (Halobacteriales) accumulate KCl to molar concentrations to counterbalance the external salinity 2324252627. Although almost nothing is known about the mechanism of KCl accumulation in Halanaerobiales, the process is well understood in Halobacteriales. The electrical potential (Δψ) that drives the uptake of potassium ions in these organisms results from the concerted action of the membrane bound proton-pump bacteriorhodopsin and the "proton gradient-consuming" proteins ATP synthase and Na⁺/H⁺ antiporter 28. Potassium is taken up via a K⁺ uniport mechanism. To enable such a transport the electrical potential (Δψ) has to be greater than the diffusion potential of K⁺ (Δψ _K). The counterion chloride is taken up either by primary or secondary transporters 29. In the dark, a light-independent Cl^-/Na⁺ symporter is employed 30, but only little is known so far about this transport mechanism and the transporter is unknown. A far better understood transport of Cl^- is accomplished via the light-driven chloride pump halorhodopsin 31. Halorhodopsin is a heptahelical membrane protein and together with bacteriorhodopsin and sensory rhodopsins belongs to the subfamily of archaeal rhodopsins 3233. To its only lysine residue (Lys²⁴²) a retinal is bound covalently as a protonated Schiff base 33. This Schiff base, together with the surrounding amino acids, forms the active centre of the chloride pump, which is able to absorb green light with a maximal wavelength of 578 nm. The absorption of one photon triggers a photochemical reaction that leads from all-trans to a 13-cis configuration. This isomerization reaction is coupled to the transport of one molecule of Cl^- from the surrounding medium into the cell 3435.

To date, only one representative of the aerobic bacteria is known that also shares the strategy of KCl accumulation to cope with extreme salinities. The extremely halophilic Salinibacter ruber was isolated from saltern crystallizer ponds in Spain 36 and was reported to need a minimal salt concentration of 1.7 M to allow growth. The optimal salinity was found between 2.5 and 3.9 M NaCl. It accumulates very high concentrations of Cl^- and K⁺/Na⁺, while other solutes were found only in minor concentrations 37. Although biochemical analyses addressing the uptake of K⁺ and Cl^- are not yet available, it was concluded from genome analyses that potassium could be taken up via a TrkHA transport mechanism. S. ruber possesses 4 copies of a putative trkA and two copies of a putative trkH gene that seem to originate from different sources via lateral gene transfer 38. TrkH is the membrane spanning translocating subunit and TrkA is the cytoplasmic membrane surface protein that binds NADH/NAD⁺ and which is essential for the transport activity 39. Similar to the halobacteria, chloride could be taken up by the means of the chloride pump halorhodopsin. Altogether 4 putative genes encoding a rhodopsin were identified in the genome. Two of which were, based on sequence similarity, assigned to be sensory rhodopsins, one to be a proton pump and one a chloride pump 38. Additionally, two copies of Na-K-Cl cotransporter – genes were identified that are common in eukaryotes but seldom found in prokaryotes. The physiological role of such a transporter is unknown for prokaryotes, but eventually contributes to the accumulation of K⁺ and Cl^- in S. ruber cells 38.

As a prerequisite for this so called "salt-in-cytoplasm" strategy these organisms have a highly adapted, salt-tolerant or even salt-dependent enzyme equipment, that shows a high amount of acidic and a low amount of aromatic amino acid residues compared to non halophilic organisms 40234142. This adaptation, however, sets clear restrictions on the habitat in which these organisms can exist, since the proteins always need a quite high intracellular salt concentration for correct protein folding and activity 24. However, it should be mentioned that this is rather a tendency than a rule. It was reported for Haloferax volcanii (Halobacteriales), for example, that this organism is able to adapt to salinities between 1 M and almost saturation 4344, but nothing is known about the mechanism of osmoregulation in H. volcanii.

The moderately, halophilic bacterium Halobacillus halophilus was originally isolated from salt-marshes at the North Sea coast of Germany. It is an aerobic, rod-shaped, motile, and endospore-forming Gram-positive bacterium of the low GC-branch that was initially classified as Sporosarcina halophila 45. Based on 16S rRNA data analysis it was later reclassified as Halobacillus halophilus 46. This organism has optimal growth rates between 0.5 and 2.0 M NaCl and was reported already in its first description to grow in the presence of NaCl but not Na₂SO₄. This first observation for a crucial role of chloride in Halobacillus halophilus was later confirmed by more detailed analyses 47. It was shown that a minimal Cl^- concentration of 0.2 M is needed to support cell growth and that, with exception of bromide, no other halide was able to substitute chloride in this respect. After prolonged adaptation of Halobacillus halophilus to nitrate, it was also able to support growth, but growth rates as well as final optical densities were lower than observed for chloride. However, it is worthwhile to note that bromide as well as nitrate is of no physiological relevance, since the concentrations used in the growth experiments are not found in nature. Additionally, the intracellular chloride content of the cells was measured. The internal Cl^- concentration was clearly related to the external concentration in the medium. At low Cl^-_e, Cl^- was largely excluded from the cytoplasm. Increasing Cl^-_e lead to increasing Cl^-_i until a steady state was reached at 0.5 – 0.8 M Cl^-_e. At that concentration, the internal concentration was found to be about half the external concentration, leading to the first idea of chloride serving as a solute in Halobacillus halophilus 47 (Fig. 1). This hypothesis was questioned when later investigations revealed that chloride does not only support growth but also is essential for motility and endospore germination 4849. These observations pointed to a more global role of chloride in the physiology of Halobacillus halophilus. To address this question the effect of Cl^- on motility was analyzed. Biochemical and molecular data proved that transcription of fliC, encoding the structural protein of the flagellum, as well as flagellin production were strictly dependent on chloride and these studies gave rise to the hypothesis that chloride could serve as a newly discovered signal molecule in Halobacillus halophilus 50.

A comparison of the proteome of nitrate and chloride grown cells revealed 5 additional proteins that were specifically produced in the presence of chloride. Among those proteins, which were tentatively identified by BLAST searches, members of the general stress regulon, as described for B. subtilis 5152, were found. One was SodA, a superoxide dismutase, another YvyD, a modulator of the sigma factor σ^L. Furthermore, an N-acetylmuramoyl-L-alanine amidase that is essential in cell wall biosynthesis, a protein with unknown function and a protein (LuxS) that is essential in the production of autoinducer-2 or in the activated methyl cycle were found 50. For the latter the proteome data were confirmed by additional studies, which not only revealed a chloride dependent production of LuxS, but also showed a clear influence of chloride on luxS transcription 53. In sum, the data obtained from proteome analysis suggest the existence of a global regulatory network of processes that are dependent or at least are strongly influenced by chloride 54.

Since Cl^- is indispensable for growth it has to influence a growth essential process. The nature of this process was unidentified for a long time. For a moderate halophile a definitely essential function is to measure external salinities and to adapt to changing osmolarities in the habitat. Halobacillus halophilus, as already mentioned, was isolated from salt marshes. The salt marshes represent an elevated, coastal area that is flooded during high tides with sea water that possesses an average salt concentration in the North Sea of about 3.5%. The salt is mainly NaCl. Upon evaporation and additional salt agglomeration by the sea wind the salt concentrations can rise up to 10% in the salt marshes, but drop almost to the level of freshwater after rainfall. The first hint that chloride could be involved in osmoregulation and the accumulation of compatible solutes came with investigations published by Roeßler and Müller in 2001. Transport studies with radiolabelled glycine betaine revealed a strict sodium and chloride dependence of glycine betaine uptake as response to osmotic upshock. Inhibitor studies suggested a primary transport system 55. In the absence of glycine betaine, Halobacillus halophilus copes with the effect of external salt by accumulating of a cocktail of different compatible solutes, mainly amino acids such as glutamine, glutamate, proline and alanine but also of amino acid derivatives such as N^ε-acetyl lysine, N^δ-acetyl ornithine, glycine betaine and ectoine 56. The obvious question that arose from these considerations was whether the accumulation of compatible solutes via de novo biosynthesis was also a chloride-dependent process and whether this was the key to an explanation for the chloride dependence of Halobacillus halophilus. To address this question intensive biochemical and physiological studies were done to solve the pathways for compatible solute biosynthesis, to solve their regulation and to clarify the role of chloride in these processes.

The finding that chloride is essential for osmoprotection and the accumulation of the compatible solutes glutamine, glutamate, proline and ectoine extended the model of a chloride dependent regulatory network in Halobacillus halophilus. It was shown that chloride affects the accumulation of these compatible solutes very dramatically and acts as a regulator on different levels, namely transcription, translation, but also enzyme activity (Fig. 8). Based on these results, a reasonable explanation for the essential role of chloride in Halobacillus halophilus was given for the first time.

Concomitantly with the identification of the growth-essential process of osmoregulation as a chloride dependent process, the question about the molecular basis for chloride as a regulator molecule in this process comes up. In this context one has to keep in mind that before solutes are accumulated as a response to hyperosmolarity the cell has to sense osmolarity somehow. The process of sensing osmolarity is generally only poorly understood and almost not investigated in moderate halophiles. Studies with the halotolerant model organism E. coli lead to the assumption that the initial signal that is sensed after an osmotic upshock is the reduced turgor pressure that establishes due to a loss of water. This reduced turgor leads to an influx of K⁺ within seconds via the potassium transporters Kup, Trk, Kdp 7374 but also activates the KdpD-KdpE regulatory system which in turn regulates the expression of the kdpFABC operon encoding the Kdp transport system. The KdpD-KdpE system is a classical, bacterial regulatory two-component system with KdpD being the membrane-bound sensor kinase and KdpE being the response regulator. The actual mode of sensing the turgor pressure that leads to the autophosphorylation of KdpD is still a matter of debate, but was postulated to be due to alterations of the transmembrane helix-helix interactions, but additionally seems to be influenced by the K⁺ concentration 757629.

Contrary to this model, the osmosensing two-component system MtrB-MtrA of Corynebacterium glutamicum does not respond to a reduced turgor pressure after osmotic upshock. Instead, the addition of different chemical compounds, like sugars, amino acids or polyethylene glycols seems to trigger the autophosphorylation activity of MtrB. The proposed explanation for this is the change in the hydration state of the protein caused by the solutes 77. In Halobacillus halophilus two-component systems are present, but none of them could definitely be assigned to be of the KdpDE or the MtrBA type. However, the actual signal that is recognized as well as the mechanism might be different in different organisms.

However, not only two-component systems may serve as potential osmo-sensors, but also transporter proteins. BetP of C. glutamicum or E. coli is a Na⁺/glycine betaine symporter of the betaine-carnitine-choline transporter family (BCCT) that is strongly regulated by the osmolality of the medium. The regulatory domain was shown to be located in the C-terminus that extends into the cytoplasm. Due to its basic characteristics it is believed to interact with the cytoplasmic membrane, but loses its interaction upon K⁺ accumulation after osmotic upshock. The loss of interaction in turn leads to a conformational change within the protein and to an increase of specificity for its substrate glycine betaine 787980.

A similar mechanism of "sensing" the surface charge of the membrane was proposed for the ATP-hydrolyzing ATP-transporter OpuA of Lactococcus lactis. OpuA is a high-affinity transporter for glycine betaine that effectively can be switched on or off by osmotic upshift or downshift, respectively 81. The molecular sensors with which the osmotic strength can be measured were identified to be the C-terminal CBS domains of the ATP-hydrolyzing subunits. They possess a very anionic character and were therefore postulated to be influenced by the negatively charged membrane. Similar to BetP the change of the ionic strength could result in a conformational change and therefore activation of the transporter 82.

In contrast to the enteric bacteria, like E. coli for example, or the Gram-positive bacterium C. glutamicum, that have to be able to respond to fluctuating osmolality rather than salinity, the major osmolyte Halobacillus halophilus is confronted with, is NaCl. It would therefore be an attractive explanation that Halobacillus halophilus senses changing salinities by sensing changing chloride concentrations. The actual mechanism is unclear to date, but several models are conceivable: (i) Chloride could be measured via an extracellular receptor. The binding of substrate would then trigger a signal transduction cascade into the cell similar to a two component system. The disadvantage of this model would be that it cannot explain how the intracellular osmotic state is measured. (ii) Another concept could be based on the idea that changes in salinity could be measured via a chloride transport process into the cell. The import could be measured by a specific transporter or by an associated protein which then leads to a signal transduction cascade and finally to a cellular response. A similar model was already proposed for the moderately halophilic bacterium Halomonas elongata where the transport of the compatible solute ectoine is thought to be the osmosensor 83. (iii) A chloride-binding regulator in the cytoplasm could directly interact with the anion and could, depending on the chloride concentration, trigger cellular responses. One putative candidate for such a function is the regulator protein YvyD that was identified in chloride-but not in nitrate-grown Halobacillus halophilus cells 50. The latter two models have the advantage that they provide a possible mechanism, with which changes in the external salinity – that are reflected in the intracellular chloride concentrations – can be measured. However, the fact that very different processes are affected by the presence or absence of chloride in Halobacillus halophilus demands the presence of a global regulatory system. The crucial point of these concepts is that they cannot be explained by the presence of only one regulatory protein, since no regulator is known to date that is able to regulate on such different targets as transcription, translation and enzyme activity at the same time. In Halobacillus halophilus no direct evidence was found so far, with exception of YvyD, that a multi component regulatory network of chloride dependent regulators exists.

The regulating mechanisms listed above require specific ion-target interactions and are functional already at very low ion concentrations. The functionality and specificity is based mainly on the chemical properties of the specific ion. But with increasing ion concentrations the physical effects of the salt solution becomes more pronounced and unspecific interactions (direct or indirect) become important. Among the effects that are caused by high salt concentrations are (i) the depletion of water molecules, (ii) a change in water activity, (iii) osmotic activity or (iv) hydrophobic interactions. A conceivable model for sensing osmolarity, therefore, would also imply the measurement of such physical parameters by cellular compounds. The effect of inorganic ions on biological matter was investigated already at the end of the 19^th century. Franz Hofmeister detected that especially anions have the capacity to precipitate proteins (globuline) out of solutions. The capacity to do so is different for different anions. Based on this observation he was able to rank the anions in a series that became known as the "Hofmeister series" (Fig. 9). In this series sulfate had the strongest influence on precipitation followed by phosphate, acetate, citrate, tartrate, bicarbonate, chromate, chloride, nitrate and chlorate 84. Centuries later the exact molecular basis for the Hofmeister series is still enigmatic, but it becomes more and more obvious that it is due to direct ion-macromolecule interactions and interactions of ions with water molecules in the first hydration shell of the macromolecule. Thereby, kosmotropic ions are differentiated from chaotropic ions. Kosmotropic ions are strongly hydrated, act stabilizing on macromolecules and show a salting-out effect. In contrast, the chaotropic ions directly interact with macromolecular surfaces and destabilize folded proteins. They exhibit a salting-in behavior [for review and further reading see [85] 86. The relevance of the Hofmeister series in biological systems was demonstrated by several studies that focused on enzyme activity 878889, on protein stability 9091, on protein-protein interactions 9293 or on bacterial growth 9495.

Based on the Hofmeister series and the effects of anions on proteins or whole cells, a different concept can be proposed for Halobacillus halophilus to measure osmolarity. Halobacillus halophilus is a moderately halophilic organism that has adapted to saline environments. Since chloride is not excluded from the cytoplasm but rather accumulates, depending on the external salinity, to molar concentrations within the cell 47, it is principally thinkable that the physical effects caused by the presence of chloride is of regulatory importance. A prerequisite for the usage of such a strategy is the evolutionary development of proteins that are functional in the presence of such high ion concentrations. As a consequence such enzymes might be dependent on the presence of minimal Cl^- concentrations for optimal functionality. Examples for such a behavior were already found in Halobacillus halophilus in the case of the glutamine synthetase that demands an optimal salinity of 1.5 M NaCl 58. Such an adaptation to high salinities is already observed in extremely halophilic organisms such as members of the Halobacteriaceae or Salinibacter ruber and it would be in good correlation with the identification of 7 positively charged residues in the deduced protein sequence of GlnA2 – the postulated chloride dependent glutamine synthetase – at which chloride could bind 58. The Hofmeister series not only offers an explanation of the regulatory influence of chloride on cell metabolism in Halobacillus halophilus but also offers an explanation to the regulatory influence of glutamate in the process of pro-gene transcription. Under physiological pH glutamic acid is deprotonated to the anion glutamate^- that accumulates within the cell to molar concentrations. Since too high concentrations of such anions are inhibitory for cell metabolism, the cell has to substitute glutamate^- by an electroneutral compatible solute like proline. The same mechanism was previously proposed for E. coli that substitutes glutamate^- by trehalose 16. Again, the cell has to "sense" the concentration of an anion (in this case glutamate^-). For Halobacillus halophilus it was shown that a minimal external glutamate concentration of 0.2 M is sufficient to stimulate pro gene transcription very effectively 60. Following these data it was speculated that not (only) NaCl is the initial signal that triggers the production of proline, but also the internal glutamate concentration, that increases with increasing salinity. Glutamate is therefore regarded as a "second messenger" in Halobacillus halophilus beside of being a compatible solute. This idea was nicely corroborated by studies done in the group of Prof. Gralla that demonstrate the potential of glutamate as an activator or inhibitor of transcription 9697. In sum, based on the Hofmeister series a very sophisticated model of regulatory sequences can be proposed for the osmoregulation in Halobacillus halophilus that is solely based on physico-chemical properties of anions.

A still open question is why Halobacillus halophilus additionally accumulates compatible solutes to molar concentrations although there are high concentrations of chloride present in the cell (Fig. 1). The advantage of such a hybrid strategy (salt-in and compatible solute in) lies at hand. Strictly halophilic archaea are dependent on molar concentrations of NaCl in their environment and exclusively use the salt-in strategy to counterbalance the low water activity. Although single members of the haloarchaea are known to be able to grow over a broad range of salinity (H. volcanii for example), they still depend on minimum NaCl concentration of 1.5 M or higher to grow. This dependence is due to a very pronounced adaptation of the whole enzymatic machinery on high salt concentrations. Such a radical adaptation would be counterproductive for a moderate halophile like Halobacillus halophilus thriving in habitats where salinities temporarily can reach molar concentrations but also can fall to freshwater concentrations after rainfalls. Instead, an only modest adaptation to elevated salinities on the protein level would be advantageous. It could help the cell to tolerate a sudden or dramatic increase of salinity that concomitantly would result at least in a temporary increase of salt concentration in the cytoplasm, easier, since enzymes are still active. However, such an adaptation on the enzyme level requires the presence of a minimal salinity outside and within the cell for optimal operation and could therefore explain the chloride concentrations measured in Halobacillus halophilus. The nature of the cation that balances the negative charge of chloride is not yet known, but could be K⁺. An only modest adaptation to elevated salinities within the cell also explains the need for alternative osmoprotectants at high external salinities. Therefore, Halobacillus halophilus has to accumulate compatible solutes such as glycine betaine, glutamate, glutamine, proline or ectoine in addition.

In summary, a dual strategy in osmoprotection is able to explain the broad range of tolerated salt concentrations that is common to all moderately halophilic organisms. In the context of this model Halobacillus halophilus represents an intermediate in respect to its demands of NaCl between the non halophilic and the extreme halophilic organisms. It is therefore conceivable that this organism employs an intermediate strategy of salt-in-cytoplasm and the use of compatible solutes.

The model of Halobacillus halophilus as an organism that uses an intermediate strategy for osmoregulation and physico-chemical properties of anions to regulate the processes of compatible solute accumulation opens a whole new field of regulatory mechanisms to study. Future investigations will have to show for example whether chloride directly influences the activity of the glutamine synthetase or if the stimulation of activity is mediated by a so far unknown factor. Further, the role of glutamate will have to be investigated in more detail. For this purpose in vitro experiments have to be established to study its effect on transcription. Also the role of ectoine will be an issue for further investigations. Can ectoine production also be triggered by other stimuli than salinity? And what then happens in an ectoine-deficient mutant? The answer at least to the last question demands the availability of a genetic system that hopefully will be at hand soon.