Stress responses of Bacillus subtilis to high osmolarity environments: Uptake and synthesis of osmoprotectants

BETTINA KEMPF and ERHARD BREMER* Department of Biology, Laboratory for Microbiology, Philipps University Marburg, Kart-von-Frisch Str, D-35032 Marburg, Germany *Corresponding author (Fax, 49-6421-288979; Email, [email protected]).

A decrease in the water content of the soil imposes a considerable stress on the soil-living bacterium Bacillus subtifis: water exits from the cetts, resulting in decreased turgor and cessation of growth. Under these adverse circumstances, B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated via de novo synthesis, but B. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute of B. subtitis is the potent osmoprotectant gIycine betaine. This trimethylammonium compound can be taken up by the ceil through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment, B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence of B. subtitis against high osmolarity stress.

1. Introduction envelope of microorganisms is permeable to water, fluc- tuations in the environmental osmoIarity trigger the flux The osmotic strength of the environment is an important of water across the cytoplasmic membrane along the physical parameter that influences the ability of micro- osmotic gradient. To avoid lysis under low-osmolarity organisms to proliferate and successfully compete for a or dehydration under high-osmolarity growth conditions, given habitat. Bacteria maintain an osmotic pressure in bacteria must possess active mechanisms to efficiently the cytoplasm that is higher than that in the surrounding adjust the osmolarity of their cytoplasm. environment, resulting in an outward directed tension, AnaIyses of the cellular responses of bacteria to osmotic the turgot (figure 1). In Gram-negative bacteria, turgor stress have been focused primarily on high-osmolarity has been estimated at 0.3-0.5MPa (Csonka 1989), environments (Csonka 1989; Csonka and Hanson 1991; whereas Gram-positive bacteria, such as Bacillus subtilis, Galinski and Trtiper 1994; Lucht and Bremer 1994; maintain a much higher turgot, estimated at approximately Csonka and Epstein I996; Miller and Wood 1996). Two 1.9MPa (Whatmore and Reed 1990). Since the cell basic schemes of adaptation to these environments have

Keywords. Stress responses; osmoprotection; compatible solutes; osmoregulation; transporters

J. Biosci., 23, No. 4, October 1998, pp 447--455. Indian Academy of Sciences 447 448 Bettina Kempf and Erhard BPemer

glycosylglycerot), quaternary amines and their sulphonium analogues (e.g., glycine betaine, proline betaine, dimethyl- sulphoniopropionate), sulphate esters (e.g., choline-O- sulphate), N-acetylated diamino acids (e.g., N6-acetyl- H2o ornithine, Ne-acetyllysine), and small peptides (e.g., N-acetylglutaminylglutamine amide) (Csonka 1989; Galinski and Trfiper 1994). In general, compatible solutes are highly Soluble molecules and do not carry a net charge at physiologicat pH. In contrast to inorganic salts, they can reach high intracellular concentrations without disturbing vital functions such as DNA-replication, DNA- protein interactions, and cellulhr metabolism. high Compatible solutes serve a cluaI function in osmo- H20 = osmolarity regulating cel[s. Because they can accumulate up to molar concentrations, they make a major contribution to the osmotic balance of the cytoplasm (Galinski and Trtiper 1994; Miller and Wood 1996). They also serve as stabilizers of enzymes and cell components against

11 the denaturing effects of high ionic strength and hence can be described as chemical chaperones. This protective t property of compatible solutes appears to stem from their preferential exclusion from the hydration shell of H20 " " Turgor--~ Adaptation macromolecules (Potts 1994; Yancey 1994). In this over- 9 9 9 +.,, view, we focus on the physiological and molecular stress responses of B. subtilis to high osmolarity environments i and report on the characteristics of the systems for the Figure 1. Initial adaptation of B. st+btitis to high osmolarity. synthesis and uptake of osmoprotectants and compatible solutes in this soil bacterium. been identified: (i) The accumulation of very high 3. B+ subtilis as a model system for studies on intracelluIar concentrations of ions. This s:rategy is osmoregtflafion followed by many halophiIic and halotolerant micro- B. svbtitis frequently occupies the upper layers of the organisms whose entire physiology has been adapted to soil, where desiccation often causes high salinity and a permanent life in a high-saline environment. (it) The intracellular accumulation of compatible solutes, i.e., high osmolarity (Miller and Wood 1996; Potts 1994). organic osmolytes that are highly congruous with the To survive and grow in this ecological niche, B. subti/is must be able to adapt to high osmolarity stress. It structure and function of the ceil. This approach is of exhibits a behavioural response to brief increases in general importance; not only is it widely employed in osmolarity, whereby it swims away from the area of the prokaryotic world by (bacteria and archaea), but it high osmolarity (Wong et al 1995). It aIso responds is also used as a stress reaction in yeast, plant, animal, genetically with the induction of a large general stress and even human cells (Rhodes and Hanson i993; Biota- regulon (Antetmann et al 1997; Hocker et al 1996). The berg 1997; Burg et al 1997). members of this stress regulon are induced not only by salt but also by a number of growth-limiting conditions 2. Characteristics of compatible solutes and stationary phase signals; these stress proteins provide Osmoprotectants are defined as exogenous organic solutes cross-protection for a variety of environmental challenges that enhance bacterial growth in media of high osmolarity. and are thought to prepare non-growing ceils for a These substances may themselves be compatible solutes diverse range of stress conditions (Hocker et al 1996). or they may be precursor molecules that can be enzy- Expression of many members of the general stress regulon mafically converted to these compounds (Galinski and is under the control of the alternative transcription factor Traper t994; Miller and Wood 1996). The spectrum of sigma B (oz), whose activity is determined by a complex compatible solutes used by microorganisms comprise a network of regulatory proteins (Hocker et al t996; Yang limited number of compounds: amino acids and amino et at 1996). Although the addition of salt transiently acid derivatives (e.g., proline, glutamate, fl-alanine, triggers the enhanced transcription of many genes of the ectoine), sugars and polyols (e.g., trehalose, glycerol, a B regulon, sigB mutants are not at a survival disadvantage Osmoregulation in B. subtilis 449 compared with the wild type when exposed to osmotic transport systems, hut the molecular details of these shock or extreme desiccation under laboratory conditions transporters have not yet been elucidated. The influx of (Boylan et al 1993). Hence, o~ is elearIy not the master K § is essential for the recovery of turgor after upshock regulator for the osmostress response in proliferating (figure 1) and the subsequent resumption of growth B. subtilis cells. Under high osmolarity growth condi- (Whatmore et al 1990). Hence K § serves a crucial role tions, B. subtilis incites specific and highly co-ordinated in the initial cellular stress response of B. subtilis to genetic and physiological adaptation reactions to limit sudden increases in the environmental osmolarity. the loss of water from the cell and thus maintain turgot at a level suitable for growth (figure 1). At the core of this stress response is the intracellular accumulation of 4.2 Synthesis of the compatible solute proline osmoprotectants and compatible solutes (figure 2) through synthesis and uptake from the environment. Because high intracellular concentrations of K § are dele- terious for the bacterial cell, the massive accumulation of K* is an inadequate strategy for coping with proionged 4, The initial stress response to high osmolarity high osmolarity. In E. coli and S. typhimurium (Csonka and Epstein I996), the initial rapid accumulation of K § 4.1 Uptake of K + is followed by a long-term accumulation of compatible solutes such as trehalose via de novo synthesis and Potassium is abundant in nature and is the prevalent glycine betaine through uptake from the environment. celIular cation in the cytoplasm of bacteria. Studies with Natural abundance 13C-NIVIR spectroscopy studies the Gram-negative eubacteria Escherichia coli and Sal- revealed that in the absence of exogenous osmoprotec- monella typhimurium have shown that K § plays a primary tants, B. subtilis initiates a massive accumulation of role in the initial stress response of these organisms proline (figure 1) by de novo synthesis (Whatmore et (Csonka and Epstein 1996). Reed and co-workers (What- al 1990). Galinski and Trttper (1994) surveyed various more et al 1990; Whatmore and Reed 1990) have carefully Bacillus species and found that in addition to proline, analysed the influx of K* into B. subtilis subsequent to many produce the cyclic amino acid derivative ectoine sudden osmotic upshifts. A moderate osmotic upshock (figure 2) as their major organic osmolyte. Among the with 0.4 M NaCt results in a decrease in turgor (figure 1) surveyed species, B. subtilis represents a minority of and causes an increase in the cellular K + level from a proline producers that are unable to synthesize ectoine. basal value of approximately 315 mM to 650 mM within When a t?. subtifis culture grown ~n defined medium 1 h (Whatmore et aI 1990). Uptake of K + under these was subjected to a moderate osmotic upshock with 0.4 M conditions is apparently mediated by turgor-responsive NaCI the intracellular proline level increased from I6 mM to approximately 700 mM within 7 h after the osmotic challenge. The physiological importance of this remarkable increase is emphasized by the finding that

lh c~ CH~ proline represents 79% of the total free amino acid pool I. ~o of the cell under these growth conditions, whereas the

ca cP~ ~a~ concentration of its biosynthetic precursor, glutamate, is Glycine BeL~ine Gly~e Bel~lne Choline only moderately increased from 103mM to 167mM Aldehyde (Whatmore et al 1990). Because proline production is

Cl-b 0 o dependent on the prior accumulation of K § this ion might serve as a signal for increased proline synthesis Io. ~ /\ (Whatmore et al 1990). We found a direct correlation Catmltlne H H between the osmotic strength of the environment and Prnll'n~ the amount of intracellular proiine. Addition of 1-4 M NaCI to the minimal medium resulted in the production H3C-- CHh~CH ==~C.H--

B. ~ubtilis cells require an adjustment of the biosynthetic successful for the cloning of osmoprotectant uptake sys- pathway to permit production of proline even when high tems from the plant pathogen Erwinia chrysanthemi and concentrations of this substance are present in the celt. the soil bacterium Corynebacterium glutamicum as well Single amino acid substitutions in the ProB protein in (Gouesbet et al 1996; Peter et al 1996). several Gram-negative bacteria can greatly reduce the The opuE (osmoprotectant uptake) structural gene allosterie feed-back inhibition by proline (Leisinger 1996), encodes a proline transport system (figure 3) that operates resulting in proline overproduction and enhanced osmo- under high-osmolarity growth conditions (yon Blohn et tolerance (Le Rudulier et al 1982; Dandekar and Uratsu al 1997). It consists of a single component (OpuE) and 1988), One can thus readily envision that subtle modi- transports proline with high affinity. OpuE is a member fications in the properties of the biosynthetic enzymes of the sodium/solute symporter family, comprising pro- in B. subtilis might result in a proline biosynthetic teins from both prokaryotes and eukaryotes that obli- pathway refractory to high intracellular levels of proline gatorily couple substrate uptake to Na* symport. An in osmotically stressed cells. Alternatively, B. subtilis elevation of the osmolarity of the medium by either might use two distinct sets of proline biosynthetic genes ionic or non-ionic osmolytes results in a strong increase to meet the different demands of the cell for this amino in the OpuE-mediated proline uptake. Disruption of the acid under low- and high-osmolarity growth conditions. opuE gene yields a B. subtilis strain that is entirely Since proline can serve as efficient sole carbon and deficient in osmoregulated proline transport activity and nitrogen source in B. subtilis, additional cellular control is no longer protected by exogenously provided proline mechanisms are required to ensure that proline degrada- (yon Blohn et al 1997). Surprisingly, the B. subtilis tion is curtailed under higb-osmolarity growth conditions. OpuE protein displays highest similarity to the PutP proline permeases, which are used in E. coli, S. typhimurium and Staphylococcus attreus for the acquisi- 5. Uptake of osmoproteetants from the tion of proline as a carbon and nitrogen source, but not environment for osmoprotective purposes. PutP activity of E. coli and S. typhimurium is reduced when the cells are grown 5.1 OpuE: an osmotically regulated proline under high-osmolarity conditions (Wood 1988). In striking transport system contrast, OpuE activity is strongly enhanced in hypertonic media, highlighting the distinct physiological functions After exposure to osmotic stress, many plants accumulate of the PutP and OpuE proline transporters. large quantities of proline through de novo synthesis from glutamate (Peng et at 1996). Proline thus eventually reaches soil bacteria such as B. subtilis through root exudates and decaying plant material. Since exogenously provided proline is used by a wide spectrum of Gram- Choline negative and Gram-positive bacteria as an osmoprotectant (Csonka 1989; Lucht and Bremer 1994), it is not OpuB surprising- that B, subtilis can also use this amino acid for osmoprotective purposes. Transport assays with Glyeine radiolabelled proline revealed that B. subtilis possesses Betaine O~uA an osmotically stimulated uptake activity for this amino Choline acid (yon Btohn et al I997). Eetoine To facilitate the molecular analysis of transport systems ~/-Butyrohetaine OpuE GB-Aldehyde Crotonobetaine for osmoprotectants in B. subtilis, we developed an in Carnitine viva complementation strategy that uses an 13. cog mutant Choline unable either to synthesize, glycine betaine, or to acquire Proline Glycine Glyeine Betaine osmoprotectants from the medium via transport systems Betaine (Kempf and Breraer 1995). Because this strain, MKH13, Glycine cannot grow in high-osmolarity minimal media even in Betaine the presence of exogenous osmoprotectants (Haardt et OpuD al 1995), a plasmid must be provided that encodes systems for osmoprotectant uptake or production. Using MKH13, we were able to select and clone complementing l genes for glycine betaine synthesis, glycine betaine K+ uptake, and proline transport from B. subtilis (Kempf and Bremer 1995; Boch et at 1996; Kappes et al I996; Figure 3. Uptake and synthesis of osmoprotectants in B. yon Blohn et al 1997). This strategy proved to be subtilis. Osmoregutation in B. subtilis 45I

The OpuE-mediated transport of proline is strongly proliferation (Boch et at 1994). Glycine betaine is widely enhanced after an osmotic upshock. This increase in found in nature. It is employed as an osmoprotectant proline uptake is entirely dependent on de novo protein by pIants (Rhodes and Hanson I993) and is released synthesis, and hence the activity of the OpuE protein is from decomposing plant material and root exudates into not stimulated by elevated osmolarity. Mapping of the the habitat of B. subtilis. Its concentration is likely to initiation sites for the opuE transcripts by high-resolution vary considerabIy in the upper layers of the soil, and primer extension reactions revealed the presence of two B. subtitis thus requires effective mechanisms for its distinct opuE mRNA species whose amount is regulated uptake from the environment. Through gone complemen- in response to the osmolarity of the environment (yon tation and mutagenesis experiments, we identified three Blohn et al 1997). Mutant analysis demonstrated the highly effective glycine betaine transport systems in B. presence of two promoters, opuE-P1 and opuE-P2, that subtilis: OpuA, OpuC, and OpuD (figure 3) (Kempf and are recognized by the house-keeping sigma factor of B. Bremer 1995; Kappes et at 1996). Each of these is subtilis, o a, and the alternative transcription factor, tys, under osmotic control and actively participates in the respectively. The oB-dependent control of opuE-P2 activity stress reaction of B. subtitis to a high-osmolarity envi- links the OpuE transporter to the general stress regulon ronment. Osmoprotection by glycine betaine is completely of B. subtitis. Although the addition of salt triggers abolished only in a mutant that lacks all three uptake enhanced transcription of many genes of the o-~ regulon systems (Kappes et al 1996). None of these glycine (Hocker et al 1996), no clear function in osmoadaptation betaine transporters contribute to proline uptake (yon had been identified for any of its members, opuE is Bl.ohn et al 1997). thus the first example of a oS-responsive gone with a Molecular and biochemical characterization of the demonstrated physiological roIe in the osmoadaptation OpuA, OpuC, and OpuD systems reveaIed that the OpuA of B. subtitis. The activity of the opuE-P1 promoter (OpuAA, OpuAB, OpuAC) and OpuC (OpuCA, OpuCB, remains fully osmoregulated in a sigB mutant (yon Blohn OpuCC, OpuCD) transporters are mulficomponent systems et al 1997). It is apparent that two independent signal belonging to the ABC-type superfamily, whereas OpuD transduction pathways operate in B. subtilis to osmotically congists of a single component. The latter is an integral control the level of opuE expression. The opuE gone membrane protein and represents a new type of glycine might thus serve as a useful model for unravelling the betaine transport system. An OpuD-related glyeine betaine mechanism of osmosensing in B. subtilis and the pro- transporter, BetP, has recently been characterized in the cessing of this information into a genetic signal that soil bacterium C. glutamicum (Peter et al 1996). OpuD governs gone expression in response to a changing and BetP, together with the choline transporter Bert and environment. the carnitine transporter CaiT from E. coti, form a small family of structurally and functionally related uptake systems for various trimethylammoniumcompounds (Kap- 5.2 Three glycine betaine transport systems operate pes et at 1996). The OpuA and OpuC systems are related in B, subtitis to the binding-protein-dependent glycine betaine trans- porter ProU from E. cali and S. typhimurium (Lucht and Like many other Gram-negative and Gram-positive bac- Bremer 1994; Csonka and Epstein 1996; Gowrishankar teria, B. subtilis uses the trimethyIammonium compound and Manna 1996). The ProU system is characterized by glycine hetaine (figure 2) as a metabolically inert and a high-affinity (KD--l-4p.M) substrate-binding protein highly effective compatible solute (Boch et al 1994). Its (ProX), that can freely diffuse in the periplasm. As a osmoprotective capacity for osmotically stressed B. sub- Gram-positive bacterium, B. subtilis lacks this compart- tiIis cells greatly exceeds that of exogenously provided ment and the substrate binding proteins (OpuAC and proline (yon Blohn et al 1997). In B. subtitis grown in OpuCC) of the OpuA and OpuC systems are anchored minimal medium with 1 mM glycine betaine, the intra- in the cytoplasmic membrane via a lipid modification cellular level of this osmoprotectant increased after a at the amino-terminal cysteine residue to prevent their moderate upshoek with 0.4 M NaCI from approximately toss into the surrounding medium (Kempf et al 1997). 175mM to approximately 700 mM (Whatmore et at By constructing a full set of B. subtilis mutant strains 1990). Since the addition of 0.4 M salt to a basal minimal expressing or lacking particular glycine betaine uptake medium does not strongly affect the growth of B. subtitis systems, we were able to assess the kinetic parameters (Boch et al 1996), the intracellular concentration of and individual contributions made by the OpuA, OpuC, glycine betaine is likely to well surpass 700 mM under and OpuD transporters to osmoprotectant accumulation high-salinity growth conditions. This high-level accumu- (Kempf and Bremer 1995; Kappes et at 1996). OpuA lation of gIycine betaine confers a considerabte degree is clearly the dominating glycine betaine transport system of osmotic tolerance and permits the growth of B. subtitis of B. subtilis. in habitats that are otherwise strongly inhibitory for its During the analysis of subtilin production in B. subtilis, 452 Bettina Kempf and Erhard Bremer

Lin and Hanson 0995) found a chimeric proU operon intermediate (Rhodes and Hanson 1993; Galinski and encoding a multi-component transport system for glycine Trtiper 1994; Csonka and Epstein 1996). B. subtilis is betaine. This hybrid ProU system was artificially con- no exception (Bochet al 1994). Data reported in the structed by transforming a non-subtilin producing strain literature demonstrate that choline oxidation can be of B. subtilis with chromosomal DNA of a subtilin accomplished by three different enzymatic systems. In producer (Lin and Hanson 1995). The hybrid transporter the Gram-positive bacterium Arthrobacter globiformis is related to but not identical with the OpuC system and A. pascens, and the fungus CyIindrocarpon didymum characterized in our laboratory (Kappes et aI 1996, 1998). a bifunctional, soluble choline oxidase mediates glyr betaine production (Rozwadowski et al 1991). Higher 5.3 Substrate specificity of the OpuA, OpuC and plants employ a soluble choline monooxygenase to pro- OpuD transporters duce glycine betaine aldehyde and a glycine betaine A wide range of naturally occurring trimethylammonium aldehyde dehydrogenase to oxidize this intermediate into and sulphonium compounds are known to function as glycine betaine (Rathinasabapathi et al 1997). In the osmoprotectants (Csonka and Epstein 1996; Galinski and Gram-negative bacteria E. colt and Sinorhizobium meliloti Trfiper 1994). We have begun to characterize the osmo- a glycine betaine aldehyde dehydrogenase is also found; protective effects of such compounds for B. subtilis and however, in these organisms it acts in conjunction with discovered that in particular the OpuC system functions a membrane-bound FAD-containing choline dehydro- in the acquisition of a variaty of osmoprotectants from genase, which can oxidize both choline and glycine the environment. So far, we have carried out a detailed betaine aldehyde to glycine betaine at the same rate physiological and kinetic analysis of the uptake of the (Lamark et al 1991; Pocard et al 1997). carnitine, crotonobetaine, y-butyrobetaine and ectoine Our characterization of the glycine betaine synthesis (Jebbar et al 1997; Kappes and Bremer 1998). The pathway from B. subtilis revealed that this bacterium trimethylammonium compounds carnitine, crotonohetaine, employs a novel combination of enzymes (Boch et al and y-butyrobetaine are ubiquitous in nature (Kieber I996). A soluble NAD-dependent type-13I alcohol dehy- 1997) and are structurally related to glycine betaine drogenase (GbsB, glycine betaine synthesis) converts (figure 2). Kinetic and growth experiments revealed that choline into glycine betaine aldehyde, and a soluble OpuC recognizes each of these compounds with the same glycine betaine aldehyde dehydrogenase (GbsA) oxidizes high affinity (K, = 5 gM) as glycine betaine (Kappes and the aldehyde into glycine betaine. The GbsA enzyme Bremer 1998). The cyclic amino acid derivative ectoine shows striking sequence identity (between 37 and 45%) is employed by a variety of bacilli as an endogenously to the glycine betaine aldehyde dehydrogenases known synthesized compatible solute (Gaiinski and Trflper 1994). to be involved in osmoadaptation in plants and other We found that exogenously provided eetoine functioned microorganisms. However, use of a NAD-dependent type- as an osmoprotectant for the non-producer B. subtilis, III alcohol dehydrogenase in the biosynthesis of glycine and we have identified OpuC as its sole uptake route betaine has not been reported before. The B. subtilis (Jebbar et aI 1997). Osmoprotection of B. subtilis by gbsA and gbsB genes are likely to form an operon whose ectoine proved to be inefficient, which can be attributed transcription is enhanced by the presence of choline in to the low affinity of the OpuC system (K~ = 1-56 mM) the growth medium (Bochet al 1996). This observation for this compound (Jebbar et al 1997). These studies hints to the presence of a regulatory protein that mediates show the value of a genetically well characterized set the genetic control of gbsAB expression in response to of mutant strains with defined defects in osmoprotectant the availability of choline. uptake systems and revealed that the ABC transporter In osmoregulating B. subtilis cells, the glycine OpuC plays a crucial role in supplying B. subtitis with betaine-synthesizing enzymes are initiafly faced with a spectrum of osmoprotectants (figure 3). None of the substantial amounts of K* and proline, and GbsA must osmoprotectants tested thus far (with the exception of function in the presence of molar concentrations of proline) is used as sole carbon or nitrogen source by glycine betaine. To better understand the functioning of B. subtilis; therefore, these substances are accumulated these enzymes, we have begun to analyse them bio- by osmoregulating cells to serve as metabolically inert chemically. We have recently reported the characterization stress compounds. of the purified GbsA (Boch et al 1997). Although B. subtilis is not a halotolerant bacterium, the GbsA enzyme proved to be highly resistant to salt and retained 88% 6. The osmoregulatory choline glyeine betaine of its initial enzymatic activity in the presence of 2-5 synthesis pathway M KC1. The enzyme was stimulated by proline, and its In response to a water deficit, many bacterial and plant activity was only moderately inhibited by molar concen- species can synthesize g]ycine betaine from the precursor trations of glycine betaine. These features ensure that choline with glycine betaine aldehyde (figure 2) as the B. subtilis can produce high levels of the compatible Osmoregtdation in B. subtilis 453 solute glycine betaiae under conditions of high-osmolarity physiNogicaI studies on th~ release of compatible soIutes stress. in hypo-osmotically stressed cells have not yet been Uptake of the glycine betaine precursor choline is performed with B. subtitis, but stretch-activated ion chan- stimulated by the osmolarity of the environment (Both nels are present in its cytoplasmic membrane and might et al 1994), and our recent molecular analysis of choline well constitute part of the cellular protection mechanism transport in B. subtilis has established the role of two against osmotic down shocks (Zoratti et al 1990). multicomponent ABC-type transporters in choline uptake (Kappes et al 1998). One of these transporters corresponds 8. Summary and outlook to the OpuC uptake system for glycine betaine, earnit[ne, crotonobetaine and 7-butyrobetaine (figure 3), whereas Figure 3 provides a cartoon of the currently identified the second transporter, (3puB, represents a new uptake systems in B. subtilis for the uptake and synthesis of system (figure 3). Both OpuC and OpuB exhibit a high osmoprotectants and compatible solutes. The massive affinity for choline and are evolutionarity closely related. intracellular accumulation of these organic osmolytes is The structural genes for the opuC and opuB operons are an integraI part of the adaptation reaction of B. subtilis transcribed in the same direction, are located only a few to high-osmolarity stress and allows this soil bacterium kb apart on the B. sttbtilis chromosome and their gone to proliferate in habitats which are otherwise strongly products exhibit sequence identities of over 65% (Kappes inhibitory for its growth. The genes encoding these R M, Kempf B, Kneip S, Boch I, Gade J and Bremer systems for the uptake and synthesis of osmoprotectants E, in preparation). These findings suggest that the opuB thus provide a framework for the future characterization and opuC loci originate from a gone duplication event of the molecular and cellular stress responses to high with a subsequent evolution in the substrate specificity osmolarity. In particular, this will allow us to experi- of the OpuB and OpuC transporters. As detailed above, mentally address the signal transduction pathway(s) that OpuC exhibits a wide substrate specificity, whereas the allow B. subtilis to sense changes in the environmental transport activity of the OpuB system appears to be osmolarity and to identify the molecular mechanisms restricted to choline and glycine betaine aldehyde (figure converting this information into a cellular stress response. 3) (Kappes et al 1998). The unusual close structural Part of this complex network are the mechanisms that relatedness of these transporters and their different spectra allow the extrusion of compatible soIutes and ions from of osmoprotectant uptake pose intriguing questions with the celIs upon hypo osmotic shock. The complexity and respect to the molecular basis of their distinct substrate number of systems already identified for synthesis and specificity. uptake of osmoprotectants make it apparent that B. subtilis must be able to integrate them into a homeostatic network 7. Efflux of osmoprotectants of stress responses in order to survive and grow in an environment subjected to rapid changes in osmolarity. It is apparent that growth of B. subtitis in high-osmolarity environments will lead to the massive intracellutar Acknowledgments accumulation of compatible solutes either via direct uptake from the environment or through synthesis. In addition, The work of S C Lakhotia, A Tripathi, W Schumann the cell witl also contain substantial amounts of K ~ and the entire local committee at the Banaras Hindu (figure 3). However, a soil bacterium is likely 1o expe- University in Varanasi for the organization of the work- rience frequent hypo osmotic shocks caused by rain and shop is greatly appreciated. The financial travel assistance flooding. Such conditions will lead to a rapid and massive of the German Ministry of Research for EB to attend influx of water into the celh and the bacteria must be the meeting in Varanasi is gratefully acknowledged. able to quickly reduce their intracellular solute pool to Financial support in our Iaboratory on osmoregulation avoid cell lysis. Decreases in osmotic pressure are known is provided by the Deutsche Forschungsgemeinschaft to cause the efflux of K +, glycine betaine, proline, through SFB-395, the Graduiertenkolleg Enzymchemie, choline, and trehalose from E. coli and S. typhimuriurn and the Fonds tier Chemischen Industrie. cells (Csonka and Epstein I996). In the Gram-positive bacteria Lactobacillus ptantarum and C. gtutamicum, References stretch-activated channels have been detected that pref- erentially mediate the rapid efflux of compatible solutes Antelmann H, Bemhardt J, Schmid R, Much H, VOlker U and subsequent to osmotic downshock (Glaasker et al 1996; Hocker M 1997 First steps from a two-dimensional protein Ruffert et at 1997). These rapid effluxes are probably index towards a response-regulation map for Bacillus subtilis; Electrophoresis 18 t 451-1463 mediatecl in both organisms by mechano-sensitive chan- Blomberg A t997 Osmorespoasive proteins and functienaI nels and proceed in L. ptantarum by an additional assessment strategies in Saccharomyces cerevisiae; Electro- carrier-like efflux system (Glaasker et al 1996). Detailed phoresis 18 1429-1440 454 Bettina Kempf and Erhard Bremer

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