Is the glutamate the requested intracellular signal sensed by DegS kinase?

in medium supplemented with glutamate, the following question is where the glutamate comes from – is it transported from the environment or is it synthesized within the cell.

Hence, an experiment performed in medium not supplemented with glutamate would shed some light on that point.

NaCl, KCl or sucrose, the amount of the accumulated glutamate is increased. Moreover, strains with mutations in glutamate synthase or in glutamate dehydrogenase accumulated nearly normal levels of glutamate which demonstrated that none of these enzymes is solely responsible for the glutamate excess (Botsford et al., 1994). Identification of the glutamate as a counter-ion for K⁺ was reported also by Yan and co-workers. They reported that the glutamate is required to maintain the steady-state K⁺ pool in S. typhimurium (Yan et al., 1996). Glutamate accumulation in response to hyperosmotic stress was reported also for Brevibacterium lactofermentum and Corynebacterium glutamicum (Skjerdal et al., 1996).

In halophilic bacterium Halomonas elongata uptake of K⁺ was reported to occur together with synthesis of the predominant compatible solute ectoine. Also the glutamate content changes in a similar way to the K⁺ content, keeping the K⁺: glutamate ratio at a value of 2.4:1. The difference with the non-halophilic bacteria is that the potassium and glutamate levels are not replaced by compatible solutes but remain elevated at least up to 120 min (Kraegeloh and Kunte, 2002).

An interesting finding was reported recently for the halophilic bacterium Halobacillus halophilus. At intermediate salinities corresponding to 1M NaCl, cells produce glutamate and glutamine in a chloride-dependent manner (Saum et al., 2006). Besides, the bacterium can switches its osmolyte strategy and produces proline as the dominant solute at higher salinities achieved via 2 to 3M NaCl (Saum and Müller, 2007). The proline biosynthetic genes (proH, proJ and proA) form an operon which was shown to be salinity dependent with maximum at 2.5M NaCl. Chloride salts led to a highest accumulation of proline but interestingly, the chloride could be substituted to a large extent by glutamate salts. The authors analysed further these findings and could demonstrate that the mRNA levels of all three pro genes were increased up to 90-fold in the presence of glutamate. A minimal concentration of 0.2M glutamate already could stimulate that transcription. These data demonstrated that the glutamate is involved in the switch of osmolyte strategy from glutamate to proline as the dominant compatible solute during the transition from moderate to high salinity (Saum and Müller, 2007).

These results are interesting with respect to the situation in B. subtilis. It is known that after an osmotic upshock the expression of the proHJ operon, which encodes the enzymes of the biosynthetic proline production, is stimulated (Brill, 2001) and this lead to de novo synthesis of this amino acid (Whatmore et al., 1990). This biosynthetic pathway leads to accumulation of proline at the expense of glutamate and as a result, the proline content is increased to ensure the cell survival under high osmolarities, and the glutamate content is decreased (Brill,

2001). This knowledge together with the observations from the current study that the glutamate concentration is increased only within the first minutes after the salt shock reminiscent the situation in E. coli, where the initial transient accumulation of K⁺ and glutamate is later followed by trehalose production and finally replaced by compatible solutes like proline (Dinnbier, 1988). B. subtilis can not use trehalose as an osmoprotectant and the proline synthesis seem to be the second step of its osmoadaptation where it replaces the glutamate. The findings that glutamate functions as an inducer of the pro operon from H. halophilus upon high salt challenge, could be true also for the proHJ operon in B. subtilis.

Moreover up to now there is not a clue what leads to the expression of these genes. The investigations for some possible transcriptional regulators that might influence the osmotic induction of the proHJ promoter could not be identified (Dolezal, 2006).

A promising hypothesis could include the following series of events: upon elevated osmolarities B. subtilis accumulates K⁺ and glutamate as initial transient response. This increased glutamate content would lead to autophosphorylation of the DegS kinase which in turn donate the phosphor to its cognate response regulator DegU. The activated regulator then can fulfil its function by induction of the downstream genes, respectively proHJ. The expression would finally lead to the activation of ProH and ProJ, which contribute for the de novo production of the more potent osmoprotectant proline at the expense of glutamate.

Unfortunately, some of the experimental data do not support such a hypothesis – up to now the proH and proJ genes were not reported as belonging to the DegU-regulon.

Another weak point is activation of the DegS kinase through glutamate. Here, the activation through phosphorylation could be demonstrated very clearly for the DegS protein. On the other hand, the induction of the whole phosphorylation cascade including DegU was attained only in the presence of glutamate. This still support the possible role of the glutamate but does not explain the fail of the phosphotransfer in its absence, which was reported in the literature (Mukai et al., 1990; Dahl et al., 1991, 1992; Tanaka et al., 1991). Most probably it is due to a lower activity of the response regulator. All the experiments from the current study point to the fact that the glutamate could serve as an internal signal which can triggers the transduction cascade from DegS to DegU and finally transcription regulation of the respective downstream genes. There is another fact supporting the possible role of the glutamate. When the osmotic upshift is performed with cultures grown in SMM medium, the corresponding mRNA levels of degU gene revealed almost no difference compared with that under non-osmotic conditions. On the contrary, when the same measurements were performed in Helmann medium, which is used also in the current study, the accumulation of mRNA transcripts was

higher in cells subjected to high osmolarity in comparison to the low salt growth conditions (T. Hoffmann, personal communications). The observed difference in the induction levels could be attributed to the presence of glutamate in the Helmann medium and not in SMM.

However, this speculation need to be further explored since both media differ also by other components. Despite of being very good hint for the role of the glutamate, the in vitro studies with the DegS-DegU system must be further investigated. The reconstruction of the whole in vitro cascade would be necessary, so that the specific influence of the glutamate to be demonstrated for both proteins. Additionally, in vivo studies also would contribute for clarifying the role of the glutamate.

Even if one accepts that the glutamate is the intracellular signal input for the DegS kinase, there still remains one question, namely, how exactly the activities of the HK core are regulated by the presence of the glutamate.