Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

FEMSMicrobiologyLetters96 (1992) 149-154 © 1992Federationof European MicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier

149

FEMSLE05035

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coil T r o n d Lamark, Olaf B. Styrvold and A r n e R. Strcm The Norwegian Collegeof Fishery Sciences, Unirersityof TromsO, TromsO,Norway

Received 18May 1992 Revisionreceived I I June 1992 Accepted 13June 1992 Key words: Choline efflux; Glycine betaine efflux; Osmoregulation; Escherichia coil; Salmonella typhimurium; ProU

1. SUMMARY We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating ceils of Escherichia coll. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to hetaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coil, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake. 2. INTRODUCTION In osmoregulation, Escherichia coil builds up the osmotic strength of the cytoplasm by accumuCorrespondence to: T. Lam.lk, The Norwegian College of Fishery Sciences, Un'versityof Tromso, Dramsveien 291B. N-9000Tromso,Norway.

iation of K ÷ and certain organic osmolytes; e.g., glycine betaine (betaine), proline, trehalose, and glutamic acid [1-4]. E. coil obtains the highest level of osmetic tolerance by accumulation of betaine. But for betaine accumulation E. coil needs an external supply of betaine itself or its metabolic precursors choline or betaine aldehyde [5]. Synthesis of betaine from choline is mediated by the osmotically regulated Bet system, which consists of a choline dehydrogenase (BetA), a betaine aldehyde dehydrogenase (BetB), a regulatory protein (Betl), and a high-affinity uptake system for choline (BetT) with a Km value of 8 /~M [5-8]. E. coil also displays a low-affinity uptake activity for choline, but the identity of this uptake system has not been investigated previously [6]. The closely related bacterium Salmonella typhimurium lacks the Bet system [7]; but, similarly to E. coil [9], it has the ProU and ProP systems for betaine uptake [10,11]. Trehalose is the major endogenously synthesized organic osmolyte in E. coli [12]. It is accumulated in cells during growm in minimal media which lack betaine [12].

150 Studies of K + transport in E. coli have revealed that the cytoplasmic K + concentration represents an equilibrium between the rates of uptake and efflux, and both uptake [13,14] and efflux [15,16] of K + appear to be regulated at the protein level by the turgot. It has been shown previously that S. typhimurium has a ProP- and ProU-independent efflux system for proline and betaine which can be activated by sulphydryl reagents [17], and that osmoregulating cells of E. coli excrete trehalose [18]. Thus, it has become evident that there exist efflux systems for organic osmolytes, and these systems are predicted to play an important role in regulating the osmotic strength of the cytoplasm.

3. MATERIALS AND METHODS 3.1. Strains, strain constructions and growth conditions All E. coli strains used were derivatives of MC4100 (Table 1). PF6 was made by P1 transduction of proU::lacZ from BRE2071 into MC4100 and selecting for kanamycin resistance. TL722 and TL725 were made by conjugation of F ' 2 from P4X-1 into MC4100 and MHK-1, respectively, selecting for Bet + phenotype as described previously [6]. TLT00 was created by conjugation of the mutated F ' 2 plasmid of MLE33 into MHK-1, selecting for kanamycin resistance. Transductions and conjugations were performed as described by Miller [21], and the kanamycin concentration used was 60/zg/ml. The minimal medium used was medium 63 with 22 mM glucose and 0.5 or 0.7 M NaCl added [5,21]. Choline and betaine were added as indicated in the text. The cells were grown aerobically at 37°C on a rotary shaker, and growth was measured spectrophotometricaUy at 420 nm (OD4zo). 3.2. Biochemical assays To investigate choline uptake, betaine synthesis defective cells were grown in the presence of 0.5 M NaC! until the cultures reached an OD420 of 1.0. The uptake assays were then started by adding 0.5 mM p4C]-choline (specific activity 68

nCi per/,mol). At intervals, the choline content of the cells was determined by the radiochemical filtration method [6], and liquid scintillation counting in Opti-fluor scintillation liquid (Packard Instruments, Groningen, The Netherlands). To investigate choline effiux, cultures of cells which had been exposed to 0.5 mM [~4C]choline for 30 rain were devided in three parts. One part was added unlabelled betaine (0.5 mM), another part was added unlabelled choline (10 mM), and the third part was used as a control with no further additions. The choline content of the cells was determined as described above, and the bacterial growth (i.e. 0.2 generations per h) occurring during the experimental period was taken into account. Accumulation and effiux of betaine by osmoregulating cells were investigated by growing the cells in the presence of 0.7 NaCI and 0.2 or 0.5 mM [14C]-choline (specific activity 46 nCi per p.mol). At intervals, 1.0-ml samples of the cultures were centrifuged, and the top 200 p,i of the supernatant was collected. The amounts of betaine and choline in the supernatant were determined by separation on an ion-exchange column [5] and liquid scintillation counting in Hionic-fluor scintillation liquid (Packard Instrument). Since bet + strains do not accumulate detectable amounts of choline [5], the amount of accumulated betaine in these cells was calculated from the amount of labelled material which had disappeared from the growth medium [5]. The percentage of cytoplasmic betaine which was excreted from a betaine uptake-negative strain (i.e. TL725) per min, was calculated by comparing the amount of betaine appearing in the growth medium with the mean cytoplasmic betaine content during the same period. The corresponding betaine effiux from a betaine uptake-active strain (i.e. TL722) was determined by adding an excess amount (10 mM) of unlabelled betaine to the growth medium just after depletion of choline (see text) and then measuring the amount of labelled betaine appearing in the growth medium and comparing this with the mean cytoplasmic content of labelled betaine. The small amount of reaccumulated labelled betaine was neglected. Trehalose accumulated in the cells was determined by gas chro-

m a t o g r a p h i c analysis [18]. Cell p r o t e i n was d e t e r m i n e d by t h e Biuret m e t h o d as m o d i f i e d for w h o l e bacterial cells [19].

Table I Bacterial strains and phage Strain or phage

Description

Source

F - A(argF-lac)UI69 F- MC4100 proU2::lacZ osmZ F- MC4100 proU2::lacZ F - MC4100 ,~ I~aPA ) I01 A(proU )600 J( proP3 : : "In IO ) (F'2 betT + betlBA + ) (F'2 berT + betAS::lacZ)

CGSC 6152

MC4100 (F'2 berT + betA5::lacZ) MHK-I (F'2 berT + betlBA ÷ ) MC4100 (F'2 betT + betiBA + ) MHK-I

20

Wild type

C.F. Higgins

cml crl-IO0

21

E. coil

MC4100 BRE2071

4. R E S U L T S A N D D I S C U S S I O N 4.1. L o w - a f f i n i t y c h o l i n e u p t a k e

Strain MC4100 carries t h e ( a r g F - l a c ) U 1 6 9 d e l e t i o n which e n c o m p a s s e s all the bet genes, including t h e b e t T g e n e for high-affinity choline uptake. However, it has b e e n s h o w n previously that osmotically stressed cells o f MC4100 display a low-affinity u p t a k e activity for choline, with a K m value o f 1.5 m M [6]. It a p p e a r e d that actively growing cells o f t h e MC4100 derivatives M H K - I (AproU AproP AputP) and PF6 (proU::lacZ) did n o t display any d e t e c t a b l e c h o l i n e - u p t a k e activity w h e n stressed with 0.5 M NaCI a n d supplied with 0.5 m M [ lac]-choline, w h e r e a s MC4100 did (Fig. 1). Since PF6 is isogenic with MC4100 except for a A p l a c M u l 5 insertion in the p r o U

PF6 MHK-! P4X-L MLE33 TL700 TL722 TL725 s. ~'phimurium

LT2 Phage PI

19 This study E. Bremer CGSC 247

This study This study This study

CGSC. E. coli Genetic Stock Center. Yale University. New Haven. CT. Both CGSC strains were obtained from B.J. Bachmann. The term ::lacZ indicates that the gene contains a A placMu insertion.

60'

2 5O

i,o g 3o •D

20

~ ,o

i

. . . . t'+

~ x

.....-~,~ +'0

A

•

",,

...... = ......, '-i •

+'0

,'~

Time (rain)

Fig. 1. Uptake and excretion of [Z'~Cl-cholineby strains of E. coli and S. typhimurium. After 30 min (see arrows) unlabelled

choline (dotted lines: • - • ) or betaine (stippled lines: - - - ) were added. Except for after adding unlabelled choline, the accumulation of 15 dpm per/zg of cell protein corresponds to 100 nmol choline per mg of cell protein. Symbols: ra MC4100 (proU + proP + berT); Ill. MLE33 (proU + proP + betT + ); o, TL700 (proU proP berT + ); o, MHK-I (proU proP betT): A. PF6 (proU proP + betT)" and zx. S. typhimurium LT2 (proU+ proP + ).

o p e r o n , it a p p e a r s that the high-affinity b e t a i n e u p t a k e system P r o U I l l ] is identical with the low-affinity choline u p t a k e system o f E. coli. It is previously k n o w n that P r o U is an unspecific uptake system. It was originally identified as an osmotically inducible proline u p t a k e system in S. t y p h i m u r i u m [23], a n d it has recently b e e n shown that P r o U can m e d i a t e taurine t r a n s p o r t in E. coli [24]. Tiae p r o U ÷ containing strains M L E 3 3 ( b e t T + p r o U +) and MC4100 ( b e t T p r o U ~ ) had a higher capacity to accumulate choline (0.5 m M ) t h a n their p r o U d e l e t e d c o u n t e r p a r t TL700 ( b e t T + p r o U ) (Fig. 1). Since choline accumulation p e r se is previously shown not to b e a d v a n t a g e o u s to stressed E. coli cells [6], t h e P r o U - m e d i a t e d accumulation o f large a m o u n t s o f choline in M L E 3 3 and MC4100 may s e e m peculiar. However, t h e s e strains are unable to convert choline to b e t a i n e b e c a u s e o f their bet m u t a t i o n s (Table 1). It is

152 shown previously that bet ~ containing strains do not accumulate choline [5], and it is shown below that the choline content of MLE33 and MC4100 were reduced when betaine was taken up from the environment. S. typhimurium LT2 did not display any detectable choline uptake activity when assayed under the same conditions as the E. coli strains (Fig. 1). Thus, the ProU systems of E. coli and S. typhimurium appeared to have different affinities for choline. One should however keep in mind that the only function of choline in E. coli is as a precursor of betaine [5]. Since S. typhimurium lacks the Bet system and therefore is unable to convert choline to betaine, the lack of choline uptake through its ProU system may be advantageous to this organism.

4.2. Choline efflux As shown in Fig. 1, addition of unlabelled choline (10 mM) to cells containing [14C]-choline, led to a decrease in the amount of [14C]-choline in all cells tested, whereas control cells without any extra choline added displayed an increased accumulation of [~4C]-choline. It is noteworthy that the rate of decrease in the [lac]-choline content was essentially the same for the two proU + containing strains tested, whether they carried berT + (i.e. MLE33) or berT (i.e. MC4100), and that choline efflux also occurred in the proU containing strain TL700, which is betT +. These data show that, at least under conditions with a surplus of choline, choline is rapidly cycled between the medium and the internal pool of stressed E. coli cells. Furthermore, the efflux of [~4C]-choline appeared to be independent of the choline-uptake systems BetT and ProU. The addition of external betaine (0.5 raM) resulted in a decrease in the choline content of the betaine uptake positive strains MC4100 (pro + proP + betT ) and MLE33 ( proU +proP +betT +), but not of the betaine uptake negative strain TL700 (proU proP bet +) (Fig. 1). MC4100 and MLE33 lost 70% and 50% of their choline content in 15 rain, respectively (Fig. 1), and these rather high efflux rates suggest that E. coli has an efflux system for choline.

4.3. Betaine efflux From the finding that betaine uptake through ProU and ProP is not feedback inhibited by betaine, Koo et al. [17] have inferred that S. typhimurium excretes betaine during osmoregulation. In the present investigation we studied betaine efflux in actively growing cells of E. coli, by taking advantage of that bet + containing strains are able to synthesize betaine from choline. The strains used were the betaine uptake active strain TL722 (proU + proP +) and the betaine uptake negative strain TL725 (proU proP). Both strains carried the bet + genes on plasmid F'2. The medium composition was selected so that growth of E. coli depended on synthesis of betaine because of the high NaCI concentration (0.7 M), and so that the precursor [Jac]-choline (0.5 raM) was depleted before the energy source glucose. For TL725, a decrease in the choline of the culture medium was accompanied by an increase in the betaine content (Fig. 2). Evidently, betaine which was synthesized from choline, was continuously excreted from the osmoregulating cells of TL725. After choline was depleted, the concentration of betaine in the growth medium continued to rise, albeit more slowly, until it reached the same value as the choline concentration at start. Thus these cells were unable to retain betaine in the cytoplasm for an extended period. When the same experiments were performed with TL722 (proU + proP+), only a small amount of betaine was detected in the medium during the period of betaine synthesis, and no external betaine was detected after the growth medium was depleted of choline. Furthermore, TL722 displayed a higher growth yield than TL725 (Fig. 2). Presumably, betaine excreted from TL722 was rapidly taken up by its betaine uptake systems. In order to verify that betaine was also excreted from TL722 ( proU + proP +), the cells were grown under osmotic stress (0.7 M NaCI) in the presence of 0.2 mM [~4C]choline until all the choline was converted to betaine and accumulated in the cells. As shown in Fig. 3, when unlabelled betaine (10 raM) was added, the [t4C]betaine content of the growth medium increased rapidly. This finding showed that betaine was indeed cycled between the growth medium

153 and the cytoplasm of osmoregulating cells of TL722. In the experiments shown in Fig. 2, the growth rates of the two strains tested were the same before choline was depleted from the growth medium; i.e. 0.5 generations per h. However, the betaine uptake negative strain TL725 (proU proP) accumulated somewhat less betaine than did TL722 ( p r o U + proP +); i.e. 1100 and 1300 nmol betaine per mg cell protein, respectively, when determined in mid-exponential growth phase. Furthermore, the rate of choline utilization was much higher in TL725 than in TL722 (Fig. 2), and TL725 also accumulated more trehalose in the cytoplasm than did TL722; i.e. 160 "rod 40 nmol per mg cell protein, respectively. Thus, the uptake of excreted betaine through ProU and ProP led to a reduced synthesis of both betaine and trehaiose in TL722 as compared to TL725. In this study we have shown that uptake of betaine led to a net efflux of choline (Fig. 1), and this suggests a mechanism for down-regulation of betaine synthesis. Choline efflux reduces the availability of substrate for the Bet dehydrogenases, and it may also reduce the expression of the bet genes, since cytoplasmic choline is required for full derepression of the bet genes [20]. It has been reported previously that the expression of the betA a d b e r t genes [20], as well as the otsA and otsB genes for trehalose synthesis [25], is reduced by the accumulation of betaine. From Fig. 2 and Fig. 3, we calculated that the rates of betaine efflux from TL722 ( proU + prop +) and TL725 (proU proP) were about 1.6% and 1.3% per rain, respectively, of the cytoplasmic content of betaine. The similarity in the rates of betaine efflux indicates that the route of betaine efflux is the same in the two strains, and that the efflux therefore is independent of ProU and ProP. It has previously been shown that the betaine efflux system of S. typhimurium is independent of ProU and Prop [17]. We have previously suggested that E. coli has a specific efflux system for trehalose [18]. Presumably, the existencc of efflux systems for organic osmoprotectants gives the cells a high flexibility in adjusting the osmotic strength of the cytoplasm, but the osmoregulating cells of enteric bacteria have to pay for this flexibility by

l. A A 0.5 ~

y °

°°" I '0

~

a 5

&

0.50

i~"a"~

0

2

4

6

10

8

o

Time (h) Fig. 2. Kinetics of bacterial growth (o), [Z4C]cho|ine utilization (~,) and [Z4C~etaine excretion ( • ) by osmotically

stressed cells of (A) TL722 (bet" proU+ proP+) and (B) strainTL725(bet* proU proP). having to recycle betaine ([17] this study) and to resynthesize excreted trehalose which is degraded by the periplasmic trehalose [18]. However, based on a YATP value of 10 /zg dry weight of cells produced per p.mol ATP synthesized and assuming that the cells use 1 /zmol ATP per /~mol betaine transported, the energy expenditure for cycling of betaine (i.e. approx. 1.5 #.mol per dry weight per generation) amounts to only 1.5% of

i

°

i8 -

Time (h) Fig. 3. Kinetics of [laClbetaine excretion ( • ) provoked by the addition of unlabelled betaine (sec arrow), bacterial growth

(o), and [14C]choliueuptake ( zx) by osmoticallystressed cells of TL722(bet+ proU* proP* ).

154 the total energy cost o f E. coli u n d e r the p r e s e n t conditions (0.7 M NaCI).

ACKNOWLEDGEMENTS This investigation was s u p p o r t e d by g r a n t s f r o m the N o r w e g i a n Fisheries R e s e a r c h Council and the Nordic Joint C o m m i t t e e for Agricultural Research.

REFERENCES [1] Le Rudulier, D., Strom, A.R.. Dandekar, A.M., Smith, L.T. and Valentine. R.C. (1984) Science 224, 1064-1068. [2] Epstein, W. (1986) FEMS Microbiol. Rev. 39, 73-78. [3] Strom, A.R.. Falkenberg, P. and Landfald, B. (1986) FEMS Microbiol, Rev. 39, 79-86. [4] Csonka, L.N. (1989) Microbiol. Rev. 53, 121-147. [5] Landfald. B. and Strom, A.R. (1986) J, Bacteriol. 165, 849-855. [6] Styrvold, O.B., Falkenberg, P., Landfald, B., Eshoo, M.W.. Bj~arnsen, T. and A.R. Strum. (1986) J. Bacteriol. 165, 856-863. [7] Andresen, P.A.. Kaasen, I., Styrvold, O.B., Boulnois, G. and Strcm, A.R. (1988) J. Gen. Microbiol. 134, 17371746. [8] Lamark, T., Kaasen. I., Eshoo. M.W., Falkcnberg, P., McDougall, J. and Strum, A.R. (1991) Mol. Microbiol. 5, 1049-1064.

[9] May, G., Faatz, E., Villarejo, M. and Bremer, E. (1986) Mol. Gen. Genet. 205: 225-1223. [10] Cairney, J., Booth, I.R. and Higgins, C.F. ('985) J. Bacteriol. 164, 1218-1223. [11] Cairney, J., Booth, I.R. and Higgins, C.F. (1985) J. Bacteriol. 164, 1224-1232. [12] Larsen, P.L, Sydnes, L.K., Landfald, B. and Str¢m. A.R. (1987) Arch. Microbiol. 147, I-7. [13] Rhoads, D.B. and Epstein, W. (1978)J. Gen. Physiol. 72, 283-295. [14] Meury. J., Robin, A. and Kepes, A. (1985) Eur. J. Biochem. 151,613-619. [15] Bakker, E.P., Booth, 1.R., Dinnbier, U., Epstein, W. and Gajewska, A. (1987) J. Bacteriol. 169, 3743-3749. [16] Dinnbier, U., Limpinsel, E., Schmid, R. and Bakker, E.P. (1988) Arch. Microbiol. 150, 348-357. [17] KOO, S.-P., Higgins, C.F. and Booth, I.R. (1991) J. Gen. Microbiol. 137, 2617-2625. [18] Styrvold, O.B. and Strum, A.R. (1991) J. Bacteriol. 173, 1187-1192. [19] Higgins, C.F., Dorman, C.J., Stirling, D.A., Waddell, L., Booth, i.R., May, G. and Bremer, E. (1988) Cell 52, 569-584. [20] Eshoo, M.W. (1988)J. Bacteriol. 170, 5208-5215. [21] Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [22] Herbert, D., Phipps, P.J. and Strange, R.E. (1971) In: Methods in microbiology, vol. 5B. (Norris, J.R. and Ribbons, D.W., Ed.), Academic Press, London. [23] Csonka, L.N. (1983) J. Bacteriol. 151, 1433-1443. [24] McLaggan, D. and Epstein, W. (1991) FEMS Microbiol. Lett. 81, 209-214. [25] Gia~ver, H.M., Styrvold, O.B., Kaasen, I. and Strom, A.R. (1988) J. Baeteriol 170, 2841-2849.