Department of Microbiology (PUMa) Erhard Bremer Erhard Bremer (born 20.02.54) Diplom (Biology) 1980, Dr. rer. nat. (Biology) 1982, University of Tübingen, Postdoctoral fellow, National Cancer Institute, Frederick, U.S.A., 1982-84 Staff scientist (C-1), Dept. of Microbiology, University of Konstanz, 1984-90 Habilitation (Microbiology and Genetics), University of Konstanz, 1989 Assistant Professor (C-2) (Microbiology), University of Konstanz, 1990-92 Head (C-3) of the group “Osmoregulation” at the MPI, Marburg, 1992-95 Adjunct Professor (Microbiology), Department of Biology, Philipps-Universität Marburg, 1993-95; Full Professor (C-4) for Microbiology and head of the Laboratory for Molecular Microbiology, Department of Biology, Philipps-Uni- versität Marburg, 1995-current); Speaker of the DFG Collaborative Research Centre for „Soil Microbiology“ (SFB-395) (2000-2007); Vice-Dean of the Department of Biology, Philipps-Universität Marburg (since 10/2009) Synthesis and uptake of compatible solutes the osmotic potential of their cytoplasm. They rapidly as a microbial defence against osmotic and expel water-attracting solutes via membrane-embedded temperature stress mechanosensitive channels when they suddenly face hypo-osmotic conditions to reduce water entry. Hence, Microorganisms are faced in natural habitats with a cell rupture due to an undue rise in turgor is prevented. plethora of environmental challenges. Monitoring and Conversely, they accumulate ions and organic solutes adapting to changes in the environmental conditions are when they are exposed to hyper-osmotic circumstances critical processes that determine the survival of microor- to promote water retention and re-entry. Consequently, ganisms and their successful long-term competition for plasmolysis and cellular dehydration is avoided (Fig. 1). a given habitat. One of the key factors affecting micro- bial growth, both in natural settings and in the labora- Many microorganisms amass a selected group of highly tory, is the availability of free water (water activity: Aw). soluble organic compounds, the so-called compatible In particular, fl uctuations in the Aw of the environment solutes, in response to high salinity surroundings. These have profound consequences for microbial cells because solutes are fully congruous with cellular functions and such changes instigate osmotically driven water fl uxes in can be accumulated to exceeding high intracellular lev- or out of the cell. els (molar concentrations) without detrimental effects on cell physiology. Microbial cells adjust the levels of compatible solutes in a direct and sensitively graded re- sponse to the prevalent osmolarity of the environment, thereby ensuring a physiologically appropriate level of cell water and turgor. Microbial cells use both the synthesis of compatible solutes and their uptaske from environmental sources to amass these solutes under hyperosmotic stress conditions (Fig. 1). Widely used compatible solutes by Bacteria are the sugar trehalose, the trimethylammonium compound glycine betaine, the imino acid proline and the tetrahydropyrimidines ec- Fig. 1. Cellular responses of Bacilli in response to hyper- and toine and hydroxyectoine. hypo-osmotic challenges. The osmotic stress response of Bacillus subtilis As a consequence of environmentally imposed osmotic Uptake and extrusion of K+ and synthesis of the gradients, bacterial cells are threatened by rupture un- compatible solute proline in B. subtilis. We are der low or by dehydration under high osmotic condi- studying the osmotic stress response of Bacillus subtilis, tions. Bacteria cannot actively pump water across the the model organism for Gram-positive microorganisms. cytoplasmic membrane to amend their cellular hydration B. subtilis lives in the upper layers of the soil, a habitat level. Instead, they adjust their water content, and as a that is frequently subjected to changes in osmotic con- consequence their vital turgor, by actively modulating ditions caused by drying and fl ooding. Osmotic stress 148 Erhard Bremer Department of Microbiology (PUMa) elicits a set of well-coordinated genetic, physiological small DNA segment (about 120 Bp) that contains all and cellular adaptive reactions of the B. subtilis cell. DNA sequences required in cis for full osmotic control Upon a sudden imposition of salt stress, B. subtilis in- of proHJ transcription and isolated cis-active mutations duces transiently the general stress response system, that alter the relative degree of the osmotic control of a very large regulon controlled by the alternative tran- this promoter. However, the physiological and molecular scription factor SigB. Members of this regulon provide chain of events that leads to osmotic control of proHJ ex- osmotic stress resistance by physiologically not yet fully pression and the features that make this promoter osmo- understood mechanisms. The general stress response responsive remains mysterious. system is essential for adaptation of B. subtilis to an os- motic up-shock but is dispensable for adaptation of the Expulsion of compatible solutes and ions by B. B. subtilis cell to prolonged hyperosmotic stress. subtilis upon an osmotic down-shock. As outlined above, uptake of K+ and the accumulation of compatible A sudden increase in salinity triggers the uptake of K+ solutes provide a considerable degree of osmotic stress ions by B. subtilis as an immediate measure to counter- resistance to B. subtilis cells. However, the amassing of act the loss of cell water. We have previously identifi ed this very same ion and the very same solutes under high the two K+ transport systems (KtrAB and KtrCD) critical osmolarity conditions, poses a considerable threat to the for osmotic adjustment of B. subtilis. Subsequent to K+ B. subtilis cell when it is suddenly exposed to hypo-os- import, the B. subtilis cell starts to synthesise massive motic surroundings. Eventually, the stress-bearing pep- amounts of the compatible solutes proline via a dedicat- tidoglycan sacculus cannot withstand the increase in the ed osmotic stress responsive synthesis pathway (Fig. 1). intracellular hydrostatic pressure caused by water infl ux and the cell will consequently rupture. To avoid such a Concomitant with the onset of the osmo-adaptive bio- catastrophic event, both Bacteria and Archaea possess synthesis of proline, B. subtilis starts to reduce its cel- mechanosensitive channels that serve as safety vales for lular K+ content because prolonged high levels of K+ is the rapid release of organic solutes and ion. These chan- detrimental to many cellular processes. We have now nels open transiently in response to increased membrane identifi ed one of the K+ extrusion systems operating in tension and thereby form very large pores through which B. subtilis under high salinity growth conditions (Fig. 1). both ions and organic solutes can readily pass (Fig. 1). The YhaST exporter is a member of the monovalent Based on electrophysiological studies, three types of cation:proton antiporter-2 (CAP-2) family. The pheno- mechanosensitive channels (MscL, MscS and MscM) types of a yhaSTU mutant shows, however, that mul- are known to operate in microorganisms. tiple K+ extrusions systems must be present in B. subtilis that jointly function in the export of K+ subsequent to an Using B. subtilis cells that were pre-loaded with radiola- osmotic up-shift. Data from a comprehensive transcrip- bel glycine betaine under hyperosmotic conditions, we tomics study of severely salt-stressed B. subtilis cells found that osmotically down-shifted cells release com- that was conducted in collaboration with the group of patible solutes very rapidly but in a fi nely tuned fashion Prof. M. Hecker (University of Greifswald) have yielded that is directly linked to the scale of the imposed os- promising leads with respect to the nature and number motic down-shift. Based on a bio-informatics approach, of such additional K+ export systems. we identifi ed one potential MscL-type and three MscS- type (YkuT, YhdY, YfkC) channel-forming proteins in B. We have identifi ed the genes involved in the osmoregu- subtilis. We constructed a comprehensive set of single, latory proline biosynthesis and the disruption of these double, triple and quadruple mutants and tested the sur- genes (proA and proHJ) results in B. subtilis mutants vival of these mutants following a severe osmotic down- that no longer can cope effi ciently with high salinity sur- shock. The data obtained unambiguously showed that roundings. The promoter driving the expression of the the channel of large conduction (MscL) is critical for proHJ operon exhibits the strongest degree of induc- cellular survival when B. subtilis is rapidly shifted from ibility of all genes expressed at a higher level (approxi- high to low salinity conditions. The MscS-type channel mately 100 genes) in salt-stressed B. subtilis cells. We YkuT, a member of the SigB-controlled general stress have therefore focussed on a molecular characterization regulon, aids the transitions of B. subtilis from hyper- to of the proHJ regulatory region by a deletion approach, hypo-osmotic conditions. An mscL ykuT double mutant a site-directed mutagenesis study of the promoter itself does not survive a severely osmotic down-shift, empha- and a search for chromosomal regulatory mutants that sizing the role of mechanosensitive channel in the cellu- would alter proHJ expression. We have defi ned a very lar adaptation process of B. subtilis to changing osmotic 149 Department of Microbiology (PUMa) Erhard