Synthesis and Uptake of Compatible Solutes As a Microbial Defence

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 microbial 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 response 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 osmotic 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 Bremer conditions in the environment. So far, no phenotype of Salmonella typhimurium reported by Flocco and Mow- gene disruptions of the yhdY and yfkC genes was dis- bray in 1994 (J. Biol. Chem. 269, 8931-8936) cernable.

Uptake of compatible solutes via ABC transporters: structural studies of ligand-binding proteins Microorganisms can accumulate pre-formed compatible from environmental resources and can attain in this way a considerably degree of osmotic stress resistance. B. subtilis is equipped with an impressive array of uptake systems for a wide spectrum for compatible solutes, most of which are chemically related either to glycine betaine or proline. We have continued to study the substrate specifi cities and properties of transporters involved in compatible solute acquisition through physi- Fig. 2. Crystal structure of the choline/acethylcholine binding ological, biochemical and structural studies. protein EhuB from Sinorhizobium meliloti. Left: overall structure of the EhuB protein; right: details of the choline binding site. Cellular acquisition of compatible solutes by transport- The EhuB crystal structure and a molecular description of the ers poses a challenge, because compatible solutes are choline and acethylcholine ligand binding site was reported by typically preferentially excluded form the immediate hy- Oswald et al., J. Biol. Chem 390, 1163-1170, 2008). dration shell of proteins. Yet in transporters, high-affi nity and very specifi c interactions between the compatible The group of Dr. Christine Ziegler (MPI for Biophys- solute and the transport protein have to take place in or- ics, Frankfurt), in collaboration with the group of Prof. der to catalyze substrate import. Typically, Km values in Reinhard Krämer (University of Köln), reported recently the low micro-molar or even in the nano-molar range are the crystal structure of the betaine importer BetP from measured for bacterial compatible solute transporters. Corynebacterium glutamicum (Ressel et al., Nature 488, We have identifi ed ligand-binding proteins from either 47-52; 2009). BetP is a single component transporter ABC- or TRAP-transporters as readily accessible model and a member of the BCCT family of transporters de- systems to study the problem of selective interactions fi ned by our group in connection with the analysis of the between compatible solutes and proteins. In collabora- osmotically controlled OpuD glycine betaine importer of tion with the laboratories of Prof. W. Welte (University of B. subtilis (Kappes et al.; J. Bacteriol. 178, 5071-5079; Konstanz) and Prof. L. Schmitt (University of Düsseldorf) 1996). The crystal structure of the BetP protein con- we succeeded in crystallizing ligand binding proteins for tained a glycine betaine molecule serendipitously insert- the compatible solutes glycine betaine, proline betaine ed into the BetP protein during purifi cation in E. coli. and DMSA and for choline, the precursor for glycine Strikingly, the glycine betaine-binding site present in this betaine synthesis. Our crystallographic and biochemical secondary, membrane-embedded transporter is super- studies demonstrated that cation-pi interactions within imposable (Ressel et al., Nature 488, 47-52; 2009) to aromatic binding boxes of varying geometries are a cen- that discovered by us in 2004 in the soluble periplasmic tral determinant for the high-affi nity binding of these ligand binding protein ProX from the compatible solute types of compatible solutes. An example is the choline/ ABC-type uptake system ProU from E. coli (Schiefner et acethylcholine-specifi c ligand binding protein ChoX al.; J. Biol. Chem. 279, 5588-5596; 2004). Since BCCT from the soil bacterium Sinorhizobium meliloti whose type transporters and binding proteins from ABC trans- structure and ligand binding site is depicted in Fig. 2. porters are not evolutionarily related, nature has chosen In the course of these studies, we also elucidated the the very same biochemical/biophysical solution to attain crystal structure of a “closed but ligand-free” ChoX pro- high affi nity binding for a solute that is typically prefer- tein. It has been predicted in the literature that binding entially excluded from protein surfaces. proteins can adopt such a “closed ligand-free” confi guration, but only a single crystal structure of a periplasmic In close collaboration with the group of Prof. Lutz ligand-binding protein captured in such a confi guration Schmitt (University of Düsseldorf), we have focused has been described so far. This is the crystal structure on crystallographic and biochemical studies on ligand of the glucose/galactose binding protein (GGBP) from binding proteins for the compatible solutes ectoine and hydroxyectoine. These solutes are synthesized by many

150 Erhard Bremer Department of Microbiology (PUMa) members of the domain of the Bacteria and provide versa- The compatible solutes ectoine and hydroxyec- tile stress protection against osmotic challenges and both toine: versatile microbial stress protectants. Our high and low temperature stress. The crystal structure of survey of a considerable number of Bacilli revealed that the fi rst ligand binding protein of an ABC-transporter ectoine (and to a more limited extend also hydroxyec- with substrate specifi city for ectoine and hydroxyectoine toine) is widely synthesized in response to salt stress (EhuB) from the soil bacterium S. meliloti was deter- (Bursy et al., J. Biol. Chem. 282, 31147-31155, 2007) mined. We have compared the EhuB crystal structure my members of this genus. The ectoine/hydroxyectoine with that of UehA, of an ectoine and hydroxyectoine synthesis pathway has been elucidated through work in binding protein from a TRAP-type transporter present different laboratories. We have contributed to this en- in the marine bacterium Silicibacter pomeroyi. Although deavour through genetic and physiological studies in the details of the architecture of the ligand-binding site various Bacilli and by focussing in particular on the ec- found in EhuB and UehA are different, common prin- toine hydroxylase, the enzyme that converts ectoine into ciples for ligand recognition and binding of ectoine and hydroxyectoine (Fig. 4). hydroxyectoine emerged from our crystallographic studies. Indeed, guided by these principles, we were able to rationally design and construct via site directed mutagenesis “super ligand-binding variants” of the ectoine/ hydroxyectoine binding protein EhuB.

In addition to studies that focussed on multi-component transporters for ectoine and hydroxyectoine, we also discovered a single-component transport system (EctT) for these compatible solutes. The EctT transporter was identifi ed and characterized in connection with studies on osmo-stress and chill-stress protection of the soil bacterium Virgibacillus pantothenticus by exogenously provided ectoine and hydroxyectoine. The single-component ectoine and hydroxyectoine transporter EctT is a member of the above-mentioned BCCT transporter family and is related, by amino acid sequence and also by structure, to the glycine betaine importer BetP from C. glutamicum. Modelling studies conducted in collaboration with Dr. Christine Ziegler (MPI for Biophysics; Frankfurt) revealed that in all likelihood a substrate- binding site similar to that found in the ectoine/hy- Fig. 4. Surface representation of the crystal structure of the droxyectoine binding proteins EhuB and UehA (TeaA) ectoine hydroxylase from Salibacillus salexigens. The sur- is present in the BCCT-type transporter EctT. face representation of EctD shows the deep cavity housing the active site of the enzyme. The obtained crystal structure (PDB code: 3EMR) contains the iron molecule coordinated by the three amino acid residues forming the “2-His-1-carboxylate triad”. The structure of the co-factor 2-oxoglutarate is not present in the actually determined crystal structure but was modelled into the active site of EctD based on comparisons with crystal structures of different members of the non-heme iron (II)-dependent dioxygenase enzyme superfamily. The modelling study was conducted by Prof. K. Reuter (University of Marburg).

Fig. 3. EctD-mediated hydroxylation of the compatible solute ectoine

151 Department of Microbiology (PUMa) Erhard Bremer

We discovered that ectoine biosynthesis in V. pantoth- were we discovered a high affi nity TRAP-type uptake enticus is not only triggered by salt stress, as it is com- system (UehABC) for ectoines and several genes encod- monly observed in many microbial species, but also by ing enzymes for the degradation of these compounds chill stress. In contrast, heat stress does not elicit tran- for use as carbon or nitrogen sources. Expression of scription of the ectABC biosynthetic genes. These fi nd- this gene cluster (Fig. 5D) is substrate (either ectoine ings suggest that ectoine confers chill stress protection or 5-hydroxyectoine) inducible but does not respond on low-temperature grown V. pantothenticus cells. In to osmotic stress (Fig. 5B). The crystal structure of the support of this conclusion, we found that an exogenous periplasmic ligand-binding protein UehA in complex supply of either ectoine or hydroxyectoine promoted cell with ectoine was solved in collaboration with the group growth of V. pantothenticus at low temperature. We also of Prof. Lutz Schmitt. found that both ectoine and hydroxyectoine are very effective heat stress protectants for the soil bacterium Streptomyces coelicolor. Taken together, our data, and results published by other laboratories in recent years, demonstrate that compatible solutes provide stress protection to microbial cells in a way that goes well beyond the traditionally studied role of these solutes in the realm of osmotic adjustment

Although ectoine and hydroxyectoine are chemically closely related, previous in vitro and in vivo studies have suggested that these two compounds have different properties with respect to the stabilization of proteins, they inﬂ uence the melting behaviour of nucleic acids dif- ferently and physiological approaches suggest that their synthesis endows different types of stress resistance onto microbial cells. We have conducted biochemical studies Fig. 5. Utilization of ectoine and 5-hydroxyectoine as nutrients of the ectoine hydroxylase (EctD) (Fig. 4). in a marine bacterium Silicibacter pomeroyi. (A) Crystal structure of the ectoine/5-hydroxyectoine binding protein UehA in The ectoine hydroxylase is a member of the non-heme complex with ectoine [PDB code: 3FXB] (B) Substrate induced iron (II)-dependent dioxygenase enzyme superfamily. transport of ectoine in S. pomeroyi. (C) Ligand binding of UehA. The EctD ectoine hydroxylase catalyzes an enzymatic (D) Genetic organization of the ectoine/5-hydroxyectoine uptake reaction in which the O2-dependent hydroxylation of and degradation genes in the genome of S. pomeroyi. The data the substrate ectoine is accompanied by the oxidative on the crystal structure of UehA and its ligand binding proper- decarboxylation of 2-oxoglutarate to form succinate and ties were reported by Lecher et al. (J.Mol.Biol.389, 58-73)

CO2. In collaboration with Prof. K. Reuter (University of Marburg), a high-resolution crystal structure of the EctD enzyme from Salibacillus salexigens was deter- Publications mined. The solved crystal structure of EctD contains the expected iron ligand coordinated by three amino Hoffmann, T., Boiangiou, C., Moses, S., and Bremer, E. acid residues forming the so-called “2-His-1-carboxylate (2008). Responses of Bacillus subtilis to hypotonic chal- triad”. These residues and the iron ligand can be found lenges: physiological contributions of mechanosensitive in a deep, solvent exposed large cavity in the EctD pro- channels to cellular survival. Appl. Environ. Microbiol. tein in which also the co-substrate 2-oxoglutarate and 74, 2454-2460. the substrate ectoine will be bound during the catalytic cycle (Fig. 4). Horn, C., Jenewein, S., Tschapek, B., Bouschen, W., Metzger, S., Bremer, E., and Schmitt, L. (2008). Moni- In addition to being effective microbial stress pro- toring conformational changes during the catalytic cycle tectants, ectoine and 5-hydroxyectoine can also serve as of OpuAA, the ATPase subunit of the ABC-transporter nutrients and energy sources for a number of microbial OpuA from Bacillus subtilis. Biochem. J. 412, 233-244. species. We studied ectoine and 5-hydroxyectoine utilization in the marine bacterium Silicibacter pomeroyi,

152 Erhard Bremer Department of Microbiology (PUMa)

Kuhlmann, A. U., Bursy J., Gimpel S., Hoffmann, T., sis of the compatible solute 5-hydroxyectoine from Ec- and Bremer, E. (2008). Synthesis of the compatible sol- toine. (submitted) ute ectoine in Virgibacillus pantothenticus is triggered by high salinity and low growth temperature. Appl. Envi- Kuhlmann, A. U., Bursy, J., Hoffmann, T., Jebbar, M., ron. Microbiol. 74, 4560-4563. Koshy, C. Ziegler, C., and Bremer, E. EctT: a SigB-dependent BCCT-type transporter from Virgibacillus pan- Smits, S. H. J., Höing, M., Lecher, J., Jebbar, M., tothenticus for acquisition of ectoine and 5-hydroxyec- Schmitt L., and Bremer E. (2008). The compatible toine as protectants against osmotic and chill stress. solute-binding protein OpuAC from Bacillus subtilis: (submitted) ligand-binding, site directed mutagenesis and crystallographic studies. J. Bacteriol. 190, 5663-5671. Finished theses

Oswald, C., Smits, S. H. J., Bremer, E., and Schmitt, L. PhD theses (2008). Microseeding - a powerful tool for crystallizing Höing, M. (2008) Strukturelle und funktionelle Unter- proteins in complex with hydrolyzable substrates. Int. J. suchungen an Substratbindeproteinen aus ABC-Trans- Mol. Si. 9, 1131-1141. portern zur Aufnahme von kompatiblen Soluten.

Oswald, C., Smits, S.H.J., Höing, M., Sohn-Bösser, L., Opper, D. (2008) GbsR: Ein neuer transkriptioneller Dupont, L., Le Rudulier, D., Schmitt, L. and Bremer, Repressor in Bacillus subtilis zur Regulation des Cholin E. (2008). Crystal structures of the choline/acetylcho- zu Glycin Betain Biosyntheseweges. line substrate binding protein ChoX from Sinorhizobium meliloti in the liganded and unliganded closed states. J. Seibert, T.-M. (2008) Die Zellwand-Hydrolase YocH aus Biol. Chem. 283, 32848-32859. Bacillus subtilis: Genetische Kontrolle durch das essen- tielle Zwei-Komponenten System YycFG, hohe Osmo- Bursy, J., Kuhlmann, A. U., Pittelkow, M., Hartmann, larität und Kältestress. H., Jebbar, M., Pierik, A. J. and Bremer, E. (2008). Syn- thesis and uptake of the compatible solutes ectoine and Diploma theses 5-hydroxyectoine by Streptomyces coelicolor A3(2) in re- Barzantny, H. (2008) Aufnahmen und Verwertung von sponse to salt and heat stress. Appl. Environ. Microbiol. Prolin und Prolinhaltigen Oligopeptiden unter hyperos- 74, 7286-7296. molaren Umweltbedingungen in Bacillus subtilis.

Lecher, J., Pittelkow, M., Zobel, S., Bursy, J., Bönig, Bönig, T. (2008) Transposon-Mutagenese in Silicibacter T., Smits, S. H. J., Schmitt, L., and Bremer, E. (2009). pomeroyi DSS-3 und biochemische Analyse des periplas- The crystal structure of UehA in complex with ectoine matischen Bindeproteins TehA. - A comparison with other TRAP-T binding proteins. J. Mol. Biol. 389, 58-73. Ziert, Ch. (2008) Die Rolle von Bacillus subtilis yerD in der Osmoprotektion. Oswald, C., Smits, S. H. J., Höing, M., Bremer, E. and Schmitt, L. (2009). Structural analysis of the choline MSc theses binding protein ChoX in a semi-closed and ligand-free Fischer, K. (2009) Analyse zur Auswirkung der vermut- conformation. Biol. Chem. 390, 1163-1170. lichen Amidase YqiI aus Bacillus subtilis auf den Zell- wandmetabolismus von B. subtilis unter hyperosmo- Hahne, H., Mäder, U., Otto, A., Bonn, F., Steil, L., tischen Bedingungen. Bremer, E., Hecker, M., and Becher, D. (2010). A comprehensive proteomics and transcriptomics analysis of König, A. (2009) Aufnahme von Magnesium in Bacillus Bacillus subtilis salt stress adaptation. J. Bacteriol. 192, subtilis. 870-882. BSc theses Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, Bloes, D. (2008) Molekulare Charakerisierung des Sub- T., and Bremer, E. Crystal structure of the non-heme stratbindeproteins OpuCC aus Bacillus subtilis. iron(II)-dependent dioxygenase EctD mediating synthe-

153 Department of Microbiology (PUMa) Erhard Bremer

Jüngel, J. (2008) Heterologe Produktion und Aufreini- Grants gung des Substrat-Bindeproteins OpuBC aus Bacillus subtilis. Fonds der Chemischen Industrie – Bitop AG

Over, B. (2008) Die Analyse des osmotisch induzierten proHJ Promoters bei Bacillus subtilis mit Hilfe von mag- Invited lectures netischen Beads. Bundesamt für Materialforschung, Berlin (2008) Arnhold, C. (2009) Funktionelle Analyse des GbsR Re- pressors aus Bacillus subtilis. Jacobs University, Bremen (2009)

Heun, M. (2009) Funktionelle Charakterisierung der Bitop AG, Witten (2009) Xaa-Pro Dipeptidase YkvY aus Bacillus subtilis. University of Greifswald (2009) Schumacher, D. (2009) Funktionelle Analyse des GbsR Repressors aus Bacillus subtilis durch ortsgerichtete University of Maryland, College Park, USA (2009) Mutagenese. University of Würzburg (2009) Sinner, T. (2009) Funktionelle Charakterisierung der Aspartokinase aus Pseudomonas stutzeri A 1501. University of Bochum (2009)

Teich, K. (2009) Heterologe Expression und physiologis- University of Kiel (2009) che Analyse der Prolin-Iminopeptiase YclE aus Bacillus subtilis. FU Berlin (2009)

Thole, K. (2009) Physiologische Charakterisierung der BRAIN AG, Zwingenberg (2009) Xaa-Pro Dipeptidase YqhT aus Bacillus subtilis.

Weber, L. (2009) Genetische und physiologische Un- Structure of the group (12/2009) tersuchung des Peptidaufnahmesystems YclF in Bacillus subtilis. Group leader: Prof. Dr. Erhard Bremer

Ziegler, Y. (2009) Die rolle von Glycinbetain im marinen Secretary: Heike Homberger Bacillus sp. NRRL B-14911. Postdoctoral fellows: Dr. I. Budde (maternal leave), Dr. T. Hoffmann, Dr. A. Rolbetzki Knoweldge transfer PhD students: M. Höing, D. Opper, T. Seibert Cooperation with the Bitop AG (Witten-Herdecke; Ger- current: K. Fischer, M. Pittelkow many) in the fi eld of ectoine and hydroxyectoine produc- tion by microorganisms for biotechnological purposes. Diploma students: H. Barzantny, Ch. Ziert

MSc students: K. Fischer, A. König, G. Wünsche

BSc students: Ch. Arnhold, M. Heun, D. Schu- macher, T. Sinner, K. Teich, K. Thole, L. Weber, Y. Ziegler (2008/09) - A.-L. Barth, S. Henkel, L. Höcker, A. Stanek, S. Weigand, N. Widderich (2009/2010) – J. Gund (guest student from FH Mannheim)

Technical assistants: J. Gade, M. Lippmann, J. Sohn

154 Erhard Bremer Department of Microbiology (PUMa)

Awards

Teaching Award of the Dept. of Biology; Philipps-Uni- versität Marburg (to E.Bremer) (2008)

Award for best PhD-theses of the Dept. of Biology; Philipps-Universität Marburg (to M. Höing) (2008)

Award for best Bachelor-theses of the Dept. of Biology; Philipps-Universität Marburg (to M. Heun) (2009)

Address

Prof. Dr. E. Bremer Laboratorium für Mikrobiologie Fachbereich Biologie Philipps-Universität Marburg Karl-von-Frisch-Straße 8 35043 Marburg/Germany

Phone: +49 6421 282-1529 Fax: +49 6421 282-8979 E-Mail: [email protected] Bachelor students 2008/2009. Christian Arnold, Magnus Heun, Dominik Schumacher, Tatjana Sinner, Kristin Teich, Karin Thole, Lennart Weber and Yvonne Ziegler worked as Bachelor students in our group.

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