Vol. 164, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1985, p. 1224-12320021-9193/85/121224-09$02.00/0Copyright © 1985, American Society for Microbiology

Osmoregulation of Gene Expression in Salmonella typhimurium:proU Encodes an Osmotically Induced Betaine Transport System

JOHN CAIRNEY,1 IAN R. BOOTH,2 AND CHRISTOPHER F. HIGGINS'*Department of Biochemistry, University ofDundee, Dundee DDI 4HN,1 and Department of Microbiology, Marischal

College, University ofAberdeen, Aberdeen AB9 ]AS,2 Scotland

Received 17 June 1985/Accepted 22 August 1985

Previous evidence has indicated that a gene, proU, is involved in the response of bacterial cells to growth athigh osmolarity. Using Mu-mediated lacZ operon fusions we found that transcription of the proU gene ofSalmonella typhimurium is stimulated over 100-fold in response to increases in external osmolarity. Ourevidence suggests that changes in turgor pressure are responsible for these alterations in gene expression.Expression of proU is independent of the ompR gene, known to be involved in osmoregulation of porinexpression. Thus, there must be at least two distinct mechanisms by which external osmolarity can influencegene expression. We show that there are relatively few genes in the cell which are under such osmotic control.The proU gene is shown to encode a high-affinity transport system (Km = 1.3 ,M) for the osmoprotectantbetaine, which is accumulated to high concentrations in response to osmotic stress. Even when fully induced,this transport system is only able to function in medium of high osmolarity. Thus, betaine transport is regulatedby osmotic pressure at two levels: the induction of expression and by modulation of activity of the transportproteins. We have previously shown that the proP gene encodes a lower-affinity betaine transport system (J.Cairney, I. R. Booth, and C. F. Higgins, J. Bacteriol., 164:1218-1223, 1985). In proP proU strains, nosaturable betaine uptake could be detected although there was a low-level nonsaturable component at highsubstrate concentrations. Thus, S. typhimurium has two genetically distinct pathways for betaine uptake, aconstitutive low-affinity system (proP) and an osmotically induced high-affinity system (proU).

Enteric bacteria such as Escherichia coli and Salmonellatyphimurium are able to adapt to large fluctuations in theosmolarity of the environment in which they are growing.However, little is known about the molecular mechanismsby which this adaptation is achieved. In E. coli, variations inosmotic pressure are known to regulate the expression ofcertain specific genes. Thus, the relative expression of thetwo porins, OmpF and OmpC, which form diffusion chan-nels through the outer membrane can be altered by varyingthe osmotic pressure of the medium in which the cells aregrown (10-12). Expression of the kdp operon, encoding theKdp potassium transport system, also depends upon osmoticpressure. Regulation of both porn and kdp expression is atthe transcriptional level. Synthesis of membrane-derivedoligosaccharides, which are believed to maintain an osmoticbalance in the periplasmic space, is reduced by osmoticpressure (15). In this case, however, osmotic regulation isprobably not transcriptional. No other genes whose expres-sion is clearly dependent upon medium osmolarity have yetbeen identified.

It has recently been demonstrated that E. coli, S.typhimurium, and related enterobacteria can accumulateproline or betaine or both to high intracellular concentrationsin response to high osmotic pressure (1, 5, 18, 23). Prolineand betaine are known to serve as osmoprotectants in manydiverse species, including higher plants. Accumulation ofthese osmoprotectants not only restores turgor pressureacross the cell membrane but can also protect enzymes frominactivation at high ionic strength (19, 20, 28). Although theproline transport systems of S. typhimurium, and to a lesserextent those of E. coli, have been characterized in somedetail, little is known about the pathways for betaine uptakeor of the mechanisms by which transport of these com-

* Corresponding author.

pounds is regulated in response to changing osmotic pres-sure.Three genes have been implicated in proline uptake by S.

typhimurium. The major proline permease, PP-I, is encodedby the putP gene. In putP strains, some proline uptake stilloccurs via the minor permease, PP-II; this residual uptakecomponent is eliminated by mutations in the proP gene (21,24). In the accompanying paper, we have shown that PP-IItransports betaine as well as proline (1). Indeed, the kineticsof betaine uptake via PP-Il suggest that betaine, rather thanproline, is the primary physiological substrate for this tranls-port system. Thus, PP-II plays an important role in.osmoprotection by both proline and betaine (1).The third gene implicated in proline uptake is proU. In

putP proP strains, the toxic proline analog L-azetidine-2-carboxylic acid (AC) can only enter the cell and exert itstoxic effects when cells are grown at high osmotic pressure.Mutants selected as resistant to this analog at high osmoticpressure have a defect in a gene, proU, which has beenmapped to 58 min on the S. typhimurium chromosome (6). Ithas been suggested that proU encodes a third, osmoticallyinduced proline transport system, PP-Ill (6). However,proline uptake via PP-III has not been demonstrated di-rectly, and the mechanism by which transport is increased athigh osmolarity is unknown. In this paper we show thatproline uptake via PP-Ill is negligible, at least when com-pared with uptake via the other proline permeases. Rather,proU encodes a high-affinity transport system with betaineas a major substrate. Thus, S. typhimurium possesses twogenetically distinct betaine transport systems encoded byproP and proU. Expression of the proU gene is regulated atthe transcriptional level, increasing over 100-fold with in-creasing osmotic pressure. The osmotic induction of proUexpression is independent of the ompB locus, which isinvolved in the osmotic regulation of porn expression,

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TABLE 1. Bacterial strainSa

Genotype

galE503 bio-561 ompRI001::TnSbAoppBC250 leu-llSl::TnJO ompRHX0J::Tn5bgalES03 bio-561 AputPA230galE503 bio-561 AputPA230 proP1667: :Tn5galES03 bio-561 AputPA230 proPl667::Tn5 proUJ688::Mu dlgalES03 bio-561 AputPA230 proP1667::Tn5 proU1689::Mu dlgalE503 bio-561 AputPA230 proPJ667::Tn5 proUJ690::Mu dlgaIE503 bio-561 AputPA230 proPJ667::TnS proUJ691::Mu dlgalE503 bio-561 AputPA230 proPJ667::Tn5 proUJ692::Mu dlgaIE503 bio-561 AputPA230 proPl667::TnS proU1693::Mu dlgalE503 bio-561 AputPA230 proPJ667::TnS proU1694::Mu dlgalE503 bio-561 AputPA230 proP1667::Tn5 proUJ696::TnlOgalE503 bio-561 AputPA230 proP1667::TnS proUJ697::TnlOgalES03 bio-561 AputPA230 proP1667::Tn5 proUl698::TnlOgalES03 bio-561 AputPA230 proP1667::Tn5 proU1699::TnJOgalE503 bio-561 AputPA230 proPJ667::Tn5 proUI700::TnlOgaIE503 bio-561 AputPA230 proP1667::Tn5 proU1701::TnlOproUJ707::Mu dl-8proUJ704::Mu dl-8proUl705::Mu dl-8proU1706::Mu dl-8proU1704::Mu dl-8proUJ705::Mu dl-8 ompRlOOJ::Tn5bproUJ706::Mu dl-8 ompRlOOl::TrLSbproUJ702::Mu dl-8proUliO3::Mu dl-8proU1697: :TnlOgalE496 metA22 metE55 rpsL 120 hsdL6 hsdA29 Mu dl(Ap lac)Mu cts hPl no. 1

proA36 strAlpurE8 strAlpyrC7 strAIpyrF146 strAlpurF145 strAlcysCSJ9 strAlserA13 strAlcysG439 strAlcysE396 strAlzeb-609::TnlO supD1OpncA212::Mu dl-8serA15 pur-268 HfrK1O

Source or construction

29; Gibson and Higgins, submitted11This paperThis paperThis paperThis paperThis paperThis paperThis paperThis paperThis paperThis paperThis paperThis papierThis paperThis paperThis paperThis paperThis paperDonor P22 lysate CH511; recipient CH827Donor P22 lysate CH511; recipient CH828Donor P22 lysate CH511; recipient CH829This paperThis paperDonor P22 lysate CH710; recipient LT2C. L. Turnbough (7)

J. It. RothJ. R. RothJ. R. RothJ. R. RothJ. R. RothJ. R. RothJ. R. RothJ. R. RothJ. R. RothK. Hughes (13)K. Hughes (13)K. E. Sanderson

a All strains are derivatives of S. typhimurium LT2.b The ompRlOOl::TnS mutation was originally isolated as the tripeptide permease regulatory mutant tppA66::TnS (9; Gibson and Higgins, submitted).

implying the existence of at least two independent pathwaysfor the osmoregulation of gene expression.

MATERIALS AND METHODS

Bacterial strains and media. Table 1 lists the genotypes andconstruction of the bacterial strains used. All strains arederivatives of S. typhimurium LT2. Cells were grown in LBmedium (22) at 30°C (to prevent induction ofMu dl lysogens)with aeration unless otherwise stated. LC medium is LBmedium containing 2 mM CaCl2, 0.1% glucose, 0.001%thymidine, and 10 mM MgSO4. MacConkey plates were usedas described by Miller (22). Minimal glucose agar plates werebased on the E medium of Vogel and Bonner as described byRoth (25). Tetracycline, carbenicillin (an analog of ampicillin;designated as Ap), and kanamycin were used at 15, 50, and50 ,ug ml-', respectively. LOM is a low-osmolarity minimalsalts medium described previously (1).Transport assays. Transport of [14C]proline or ['4C]betaine

was assayed as described previously (1, 2). For growth atlow osmolarity, LOM-glucose was used. To induce cells for

proU expression at high osmolarity, cells were grown to an

optical density of 0.5 at 600 nm in LOM-glucose, NaCl was

added to a final concentration of 0.3 M, and the cells were

grown for a further hour. Transport assays were carried outat low osmolarity (LOM-glucose) or at high osmolarity(LOM-glucose containing 0.3 M NaCl) as described previ-ously.(1). Because of the variations in the volume of cellsgrown at different osmolarities, the amount of protein in cellextracts was determined directly, and transport rates are

expressed as nanomoles per minute per milligram of protein.Protein levels in sonicated cell extracts were determined bythe method of Bradford, using bovine serum albumin as a

standard.13-Galactosidase assays. P-Galactosidase activity in whole

cells was determined as described by Miller (22). Cells werepermeabilized by the chloroform-sodium dodecyl sulfateprocedure. Again; because the volume of cells changessignificantly with changing osmotic pressure, we determinedthe amount of protein directly during each assay, rather thanrelying on optical density as a measure of the number of cellspresent.

Strain

CH223CH511CH627CH638CH639CH640CH641CH642CH643CH644CH645CH709CH710CH711CH712CH713CH714C",826CH827CH829CH829CH831CH832CH834CH946CH947CH991JL3473

TR5656TR5657TR5658TR5660TR5663TR5665TR5666TR5667TR5668TT7610TT7674SA722

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Isolation of Mu dl and Mu dl-3 insertions. Phage Mudl(Apr lac cts62) can be used to isolate operon fusions,placing lacZ under control of any chromosomal proihoter(3). Because Mu is normally unable to infect S. typhimurium,we used a Mu-Pl hybrid helper phage which confers P1 hostrange and thus allows infection of galE strains of S.typhimurium (7). Mu dl lysates were prepared by inductionof the lysogenic strain JL3473 as described previously (1). Atleast 104 independent Ampr colonies containing Mu dlinsertions randomly distributed around the chromosomewere pooled and washed twice in minimal medium beforespreading on appropriate selective plates. Because of thepotential instability of Mu dl, strains containing Mu dlinsertions were tested genetically before each set of experi-ments to ensure that transposition had not occurred; insta-bility was never observed.Because of zygotic induction, Mu dl-mediated lac fusions

cannot easily be transduced between strains without trans-position occurring. Thus, for some experiments a recentlyconstructed, conditionally transposition-defective (andtherefore more stable) derivative of Mu dl was used (13).This phage, Mu dl-8, contains an amber mutation in thetransposase gene and is stable unless in a sup geneticbackground. Random Mu dl-8 insertions into the S.typhimurium chromosome were obtained by P22-mediatedtransduction of the Mu derivative from strain TT7674(pncA2J2::Mu dl-8) into TT7610 (supD), selecting for Amprtransductants (13). Mu dl-8 fusions initially selected andisolated in the sup strain (TT7610) were transduced into anappropriate sup' background before further characteriza-tion.

Isolation of TnlO insertions. Random TnWO insertions in theS. typhimurium chromosome were isolated by using thephage P22-TnJO system (16) as described previously (2).Once selected, TnlO insertions were transduced into a"clean" genetic background to preclude the possibility ofany secondary mutations having arisen during the construc-tion of the TnIO insertion.

Genetic techniques. Transductions were carried out with ahigh-transducing derivative of phage P22 int4 as describedby Roth (25). galE strains were grown in LB mediumsupplemented with 0.2% glucose and 0.2% galactose whenused as donors or recipients for transduction, to ensureefficient synthesis of the P22 receptors. After transduction ofTnS, TnJO, or Mu dl-8 insertions from one strain to another,the correct location of the transposon and the presence ofjust a single insertion in the transductant were verifiedphenotypically and genetically by marker rescue. All strainsused for transport and expression studies are derivatives ofCH223 and are therefore isogenic except for the introducedmutations.

Conjugations were carried out as described previously (9),using streptomycin to select against the donor HFr.Prototrophic conjugants were purified and tested on minimalglucose-tetracycline plates to determine the percentcoinheritance of the proU1697: :TnlO marker.

Identification of genotypes and phenotypes. Mutations inthe proline transport genes were identified by their patternsof resistance and sensitivity to the two toxic proline analogsAC and 3,4-dehydro-DL-proline (DHP) as described previ-ously (1, 2) (Table 2).

RESULTS

Isolation of TnlO and Mu dl insertions in proU. Mutationsin proU can be selected in a putP proP background by their

TABLE 2. Drug resistance phenotypes of proline transportmutants"

AC AC

AC DHP (40 Lg (1 mM)Genotype (40 ptg (80 F.g in the ie ten

ml) ml-) presence presenceof0.3 M NaCINaCi

putP+ proP+ proU+ s s s sputP proP+ proU+ r s s sputP proP proU' r r r sputP proP proU r r r r

aResistance (r) and sensitivity (s) were determined on minimal glucoseplates as described in Materials and Methods.

resistance to 1 mM AC on minimal glucose plates containing0.3 M NaCl (6). Thus, a pool of approximately 10,000random and independent TnWO insertions into the chromo-some of strain CH638 (AputPA proP::Tn5) was plated onminimal glucose plates containing tetracycline, 0.3 M NaCl,and 1 mM AC. ACr colonies .which appeared after 48 h werepurified and characterized further. To demonstrate that eachTetr ACr derivative contained just a single transposon inser-tion and that this transposon was responsible for the ACrphenotype, the transposon was transduced back into CH638,and each Tetr transductant was tested for resistance to AC athigh osmotic pressure. All (100% [24/24]) of such transduc-tants were ACr. One of these transductants, CH710(proUJ697: :TnIO), was used for all further experimentsdescribed here. However, six other independently derivedproU::TnIO insertions (CH709 to CH714) showed identicalphenotypes.Mu dl-mediated lacZ fusions to proU were isolated from

a pool of random insertions in strain CH638 using a selectionprocedure identical to that described above. Because ofzygotic induction, Mu dl insertions cannot be transducedinto a Mu-free genetic background. Thus, to demonstratethat each strain carried just a single Mu dl insertion and thatthis was in the proU gene, each Ampr ACr derivative wastransduced to Tetr with a P22 lysate grown on strain CH710(proUJ697::TnlO). Most (>90%) of the Tetr derivativesbecame Amps Lac-, showing that each strain contains just asingle Mu dl insertion which is very closely linked toproU::TnJO. Each of six independently isolated TnWO andseven independent Mu dl insertions in proU (CH639 toCH645) were mapped against each other in this manner, andall were found to be closely linked by transduction (>90%).Thus, all ACr mutations selected by this procedure werelocated at a single chromosomal locus, although this locusmay, of course, consist of more than one gene.Map location of TnlO and Mu dl insertions. Csonka (6)

showed that proU mutations, selected in a manner similar tothat described above, are located at 58 min on the S.typhimurium chromosome. To establish that we had selectedmutations at the same locus, the proU::TnlO insertion fromCH710 was transferred into various HFr strains, and thefrequency of cotransfer of Tetr with a number of auxotrophicmarkers was scored. These data (Table 3) show that thetransposon in strain CH710 is closely linked to cysC, thesame map location as that described by Csonka (6). Since weused a selection similar to that described by Csonka, andsince 14 independent TnWO or Mu dl insertions were alllocated at the same position on the chromosome, it seemsvery probable that mutations at only a single locus (which

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OSMOREGULATION OF GENE EXPRESSION 1227

may, of course, consist of more than one gene) can give riseto the ProU phenotype.

Osmoregulation of proU expression. It has been suggestedthat proU might encode an osmotically induced prolinetransport system (6). To determine whether this is indeed thecase and whether such regulation is transcriptional, weassayed 3-galactosidase expression from Mu dl-mediatedlacZ fusions in which P-galactosidase expression is undercontrol of the proU promoter. Figure 1 shows the expressionof 3-galactosidase as a function of salt concentration.Clearly, expression from the proU promoter increased mark-edly with increasing salt concentration. This is an osmoticeffect, rather than a specific effect of NaCl, as essentiallyidentical results were obtained if choline chloride, KCI, orsucrose was used in place of NaCl (Table 4). As controls, wehave shown that expression of Mu dl(Apr lac) fusions to anumber of other promoters (tppB, ompR, oppA, pepT) donot vary with osmotic pressure (M. M. Gibson and C. F.Higgins, submitted for publication; unpublished data). Inter-estingly, there is a threshold osmolarity below which proUinduction does not occur. Thus, the cell is able to accom-modate moderate increases in osmolarity; only under moresevere osmotic stress is the expression ofproU induced. Incontrast to other solutes, glycerol did not stimulate proUexpression. Glycerol is known to be freely permeable acrossthe cell membrane and hence would not be expected to beosmotically active. This implies that turgor pressure differ-ences across the membrane, rather than solute concentrationper se, are responsible for the induction of pro U.

Figure 2 shows the kinetics of induction of proU expres-sion. There was a slight lag of about 10 min immediately afteran increase in osmotic pressure. This was presumably due toa transient inhibition of protein synthesis caused by theosmotic shock (13). Subsequently, proU expression in-creased rapidly, reaching a maximum of about 40 min afterthe osmolarity of the medium had been increased. Theabsolute level of expression achieved is dependent upon themagnitude of the osmotic upshock.

Interestingly, betaine, and to a lesser extent proline, bothsubstantially reduced the expression of proU at highosmolarity (Table 4). No other protein amino acid had thiseffect (data not shown). This is almost certainly an indirecteffect due to the restoration of turgor pressure which resultsfrom the accumulation of these two amino acids in responseto osmotic stress.

TABLE 3. Mapping of proUa

Auxotrophic MapRecipient marker location Cotransfer

(min)

TR5656 proA36 7 0TR5657 purE8 11 2TR5658 purC7 23 12TR5660 pyrF146 33 17TR5663 purF145 47 17TR5665 cysC519 60 73TR5666 serA13 62 41TR5667 cysG639 72 21TR5668 cysE396 79 18

a The HFr strain SA722 was transduced to proU::TnJO (with CH710 astransduction donor), and this strain was used as the conjugation donor witheach of the recipients listed above. Prototrophic recombinants (100 of each)were scored for Tetr to determine the percent cotransfer of proU::TnJO witheach of the auxotrophic markers. Two other HFr's with different points oforigin were also used and gave essentially identical results.

500

Soo

400

'U

4300u-6

a-200

bc

S

0

0

a-

01 0-2 0 3 04 05NaCl concentration (M)

FIG. 1. Osmotic induction of proU expression. Strain CH946(proU::Mu dl-8) was grown in LOM-glucose containing the indi-cated concentrations of NaCl. Cells were grown to an opticaldensity at 600 nm of about 0.5, and 3-galactosidase activity wasassayed. f-Galactosidase units were calculated and expressed bythe method of Miller (22), measuring the cellular protein contentdirectly rather than using turbidity to estimate the protein content ofeach cell culture. Essentially identical results were also obtained forCH640 (proU::Mu dl).

The ompR locus is not required for osmoregulation of proUexpression. The ompR and envZ genes are involved in theosmotic pressure-dependent expression of the ompF andompC genes (10-12). To ascertain whether either of thesegenes is required for the osmoregulation ofproU expression,a Tn5 insertion in ompR, which is polar on envZ, wasintroduced into CH946 which carries a proU::IacZ operonfusion. The resultant strain (CH831) was shown to beOmpR- by its resistance to the toxic peptide alafosfalin (9;Gibson and Higgins, submitted) and to be OmpF- OmpC-by examination of the outer membrane proteins by poly-acrylamide gel electrophoresis (data not shown). The ompRmutation ompR1001::Tn5 was originally isolated as beingtripeptide transport defective (tpp) and only subsequentlywas shown to be the same gene as ompR (9; Gibson andHiggins, submitted). Eliminating ompR and envZ functionhad no effect on the expression or osmoregulation of proU(Table 4). Mutations in the other proline transport genes,putPA and proP, also had no effect on proU expression(Table 4). Although the data shown here are only for oneproU::lacZ strain, identical results were obtained for strainsCH832 and CH834.Are other genes regulated by osmolarity in a manner similar

to proU? To estimate the number of genes which are regu-lated by osmotic pressure in a manner similar to proU, weagain took advantage of lacZ fusions. lacZ fusions to proUare uninduced and therefore grow as white colonies onMacConkey-lactose plates. However, if 0.3 M NaCl is addedto the plate proU expression is induced, ,B-galactosidase isproduced by the fusion strains, and the colonies are red. Wetherefore used this screen to isolate lacZ fusions to anychromosomal gene which is osmotically induced. A pool of16,000 random and independent Mu dl-8-mediated lacZfusions to the S. typhimurium chromosome was made instrain TT7610 (supD) as described in Materials and Methods.The Mu dl-8 fusions were plated onto MacConkey-lactose

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TABLE 4. Regulation of proU expression'

Strain Relevant genotype Additives to growth medium P-Galactosidase (U)

CH946 proUJ702::Mu dl-8 None 4CH946 proUl702::Mu dl-8 0.3 M NaCl 513CH946 proUJ702::Mu dl-8 0.3 M KCI 486CH946 proUJ702::Mu dl-8 0.3 M choline chloride 328CH946 proUJ702::Mu dl-8 0.44 M sucrose 532CH946 proUJ702::Mu dl-8 0.5 M glycerol 6CH946 proUJ702::Mu dl-8 0.3 M NaCl + 1 mM proline 391CH946 proU1702::Mu dl-8 0.3 M NaCl + 1 mM betaine 180CH640 AputPA proP::Tn5 proU::Mu dl None 6CH640 AputPA proP::TnS proU::Mu dl 0.3 M NaCl 929CH831 proUJ702::Mu dl-8 ompRJOOl::TnS None 5CH831 proUJ702::Mu dl-8 ompRlOOl::TnS 529

a Cells were grown in LOM-glucose with additives as indicated, and ,B-galactosidase was assayed. ,-Galactosidase units are expressed per milligram of protein.

plates and subsequently replicated onto MacConkey-lactoseplates containing 0.3 M NaCl. Colonies which were white inthe absence of salt but red on plates to which salt had beenadded were purified and characterized further. Six indepen-dent osmotically induced fusions (each isolated from aseparate pool of insertions) were obtained by this method(CH826 to CH829, CH946, CH947). Unexpectedly, all sixwere found to be fusions to the proU gene by two criteria:each was transductionally linked (90%) to the proU::TnJO ofCH710 and each conferred ACr on high-salt plates whenintroduced by transduction into strain CH638 (putP proP).Each of the proU::Mu dl-8 fusions obtained by this proce-dure was also regulated in a manner similar to the pro U: : Mudl fusions described above. Thus, it seems that there arefew, if any, other genes whose expression is regulated byosmotic pressure in a manner similar to that of pro U.

Proline uptake through proU. Mutations in proU wereoriginally isolated on the basis of resistance to the prolineanalog AC (6). In addition, proU mutations reduced the

300

(U0

i 200

'0

bc

.

U

0--* * . 0 0

20-A 60A 10A A A

20 40 60 80 100 120

Time (min)FIG. 2. Induction of proU expression. Cells of strain CH946

(proU::Mu dl-8) were grown in LOM-glucose to an optical densityat 600 nm of about 0.5. NaCl was added to the appropriateconcentration, samples were removed after various periods ofinduction, and ,-galactosidase activity was determined. P-Galactosidase units were calculated after measuring the proteincontent of each cell culture directly. Symbols: *, 0.3 M NaCl; *,0.16 M NaCl; A, 0.06 M NaCl.

ability of proline auxotrophs to utilize exogenous proline tosupport growth. Thus, it was suggested that proU encodes athird transport system for proline, PP-III. However, prolineuptake via PP-III was never measured directly, so its realsignificance could not be assessed. We set out to measureproline uptake through PP-III and to determine whetheruptake is increased by osmotic pressure as would be pre-dicted from the expression data (see above). Figure 3 showsthe results of such an experiment. In wild-type cells (LT2)grown at low osmolarity, rapid proline uptake was observed.When a putP mutation was introduced (CH627) eliminatingPP-I, uptake was severely reduced. The residual uptake wascompletely eliminated by introducing a mutation in proP(strain CH638). Thus, strains lacking both PP-I and PP-IIshow no detectable proline uptake. Even at substrate con-centrations of up to 300 p.M proline we were unable to detectany proline uptake by a putP proP strain, showing thatPP-III plays no significant role in proline uptake, at leastunder these conditions. Because proU expression is inducedby growth at high osmotic pressure, we also determinedproline uptake for cells grown under such conditions (Fig. 3).Cells of strain CH627 (putP proP+ proU+) were grown inmedium of high osmolarity (LOM-glucose containing 0.3 MNaCl), and proline uptake was assayed at both high and lowosmolarity. As has been demonstrated previously, prolineuptake by CH627 was stimulated about threefold by growthat high osmotic pressure (1). However, all proline uptakeunder these conditions was via PP-II and was completelyabolished by mutations in proP. Even at 300 p.M prolineproU-dependent proline uptake could not be detected (datanot shown). Thus, we were completely unable to demon-strate significant proline transport through PP-III, even incells grown under conditions in which expression ofproU isfully induced. Proline is therefore unlikely to be the primarysubstrate for PP-III.proU encodes a betaine transport system. Because expres-

sion ofproU is osmotically induced, it seemed reasonable tosuppose that the gene encodes a transport system for asolute required at high osmotic pressure. Betaine seemed apossible substrate, both because it is accumulated by cells athigh osmotic pressure (1, 23) and also because, as anN-substituted amino acid, it bears at least some structuralresemblance to both proline and AC. We have shown in theaccompanying paper that the proP gene of S. typhimuriumencodes a transport system with betaine as its primarysubstrate but which also shows affinity for proline (1). Wealso found that cells have an osmotically induced betaineuptake component which is independent of proP. To deter-

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OSMOREGULATION OF GENE EXPRESSION 1229

16

14~

12

TC

0.a

0~

EC-

O.c

0=-Sai

lo0

6

41

2

x

x

0

x

--0

0 0

0 ao /LL :'km 11 2 3

TIME (mins)4 5

FIG. 3. Proline uptake through the various proline permeases.Cells of strains LT2 (putP' proP+ proU+ [xI), CH627 (putP proP+proU+ [open symbols]), or CH638 (putP proP proU+ [closedsymbols]) were grown in LOM-glucose in the presence or absence of0.3 M NaCl as indicated, and proline transport at 20 ,uM wasassayed at high or low osmolarity. Symbols: x, 0, 0, cells grownand assayed at low osmolarity; A, A, cells grown at high osmolarityand assayed at high osmolarity; *, O, cells grown at high osmolaritybut assayed at low osmolarity.

strain CH638 (proP proU+). This is in agreement with thefact that the proU gene is not expressed under such growthconditions. Transport assays on induced cells were alsocarried out in medium of low osmolarity to determine theeffects of osmolarity on the transport process itself (asdistinct from induction). Regardless of whether cells ofCH638 (proP proU+) were grown at high or at low osmolar-ity, betaine uptake could not be detected when transport wasassayed at low osmolarity. Thus, proU-dependent betaineuptake is regulated at two levels: the proU gene is onlyexpressed at high osmolarity, and in addition, once induced,the transport system will only function if the cell is underosmotic stress.

Kinetics of betaine uptake. To determine the kinetic con-stants of proU-dependent betaine uptake, we measuredtransport in strain CH638 (putP proP) at a variety of betaineconcentrations (Fig. 5). Clearly, the ProU system has highaffinity for betaine. Because of the low specific activity of the[14C]betaine it was not possible to measure transport accu-rately at concentrations below 1 ,uM. However, when theminor, pro U-independent uptake component was subtractedand kinetic constants were calculated from Eadie-Hoffsteeor Lineweaver-Burk transformations of the data in Fig. 5,the following kinetic paramaters for pro U-dependent betaineuptake were obtained: Km = 1.3 ,uM; Vmax = 12.5 nmolmin-' mg of protein-'.

DISCUSSION

When subjected to osmotic stress, bacterial cells must beable to restore turgor pressure by increasing internalosmolarity and, in addition, must protect sensitive enzymesfrom the potential deleterious effects of high intracellularionic strength. This is most frequently achieved by accumu-

80[

mine whether this transport component is dependent uponproU, we measured betaine uptake in strains variouslymutated for proP and proU (Fig. 4). Cells of CH627 (proP+proU+), CH638 (proP proU+), and CH640 (proP proU) weregrown in medium of high osmolarity (LOM-glucose contain-ing 0.3 M NaCl) to ensure full induction of proP and proUexpression, and betaine uptake was measured in medium ofhigh osmolarity. Clearly, strains mutated for proP hadreduced rates of betaine uptake; the remaining uptake com-

ponent was almost completely abolished by mutations inproU. Thus, proP and proU encode genetically independentbetaine transport systems which, between them, are respon-sible for essentially all detectable betaine uptake. At highsubstrate concentrations (300 ,uM) a low rate of residualbetaine uptake was detectable in proP proU strains (data notshown). However, this uptake component only becomessignificant at higher substrate concentrations and probablyrepresents a nonsaturable uptake pathway.The above experiments were performed with cells grown

at high osmolarity to induce proU expression. When cells ofCH627 (proP+ proU+) were grown at low osmolarity buttransport was still assayed at high osmolarity, betaine uptakewas considerably reduced (Fig. 4). Betaine uptake by cellsgrown under these conditions was wholly dependent on theproP-encoded uptake system as no uptake was detected for

T

a,

0

60cL

Ec-Sa,

=240a,a,c

A

A

0

-A

Ai/

A~~~~~

1 2 3 4 5TIME (mins)

FIG. 4. Betaine transport by proP and proU mutants. Cells ofstrain CH627 (putP [A, A]), CH638 (putP proP [0, 0, *, EU), orCH640 (putP proP proU [V]) were grown in LOM-glucose in theabsence (open symbols) or presence (closed symbols) of 0.3 MNaCl, and betaine transport was assayed at high (0, 0, A, A, V) orlow (U, L) osmolarity.

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1230 CAIRNEY ET AL.

15-{Km = 44 ,uM). In proP proU strains betaine uptake isessentially eliminated, although at high substrate concentra-tions a nonsaturable component to uptake can be detected.

E E12 E Thus, there are two active betaine transport systems in S.a) ~typhimurium. proP encodes a low-affinity uptake systemO. / 1 / which is constitutively expressed (although expression isE 9 X* slightly enhanced by increased osmotic pressure) and whichE|/ .121 <has relatively broad specificity, also transporting proline and

_X 0 /its analogs DHP and AC. proU, on the other hand, encodes.6t/j°08 a high-affinity and rather more specific transport system.

/ o Whether these two transport systems are energized byX 04 different mechanisms, as is common for different transportC3 systems for a single substrate, remains to be determined.

-.8 -6 -4 -2 *2 4 6 8 These studies were undertaken with S. typhimurium.s However, betaine can be actively accumulated by an osmot-

5 10 15 20 25 30 ically induced transport system(s) in E. coli (23). The kineticBetaine concentration parameters for uptake by E. coli are very similar to the sumBetaineconcentration(PM) of the values we obtained for the ProP and ProU systems in

FIG. 5. Kinetics of proU-dependent betaine uptake. Strain S. typhimurium. In addition, a gene equivalent to proP, atCH638 (putP proP proU+) was grown in LOM-glucose of high least in terms of proline uptake, has been identified in E. coliosmolarity (0.3 M NaCl) to fully induce proU, and betaine transport (26). Thus, it seems likely that similar betaine uptake sys-was measured at a variety of concentrations. The inset shows a tems will also be present in E. coli.Lineweaver-Burk transformation of these data. At the concentra- We showed, using Mu dl(Apr lac) fusions, that transcrip-tions of betaine used in these experiments any nonsaturable, proU- tion of the proU gene is strongly dependent upon mediumdependent uptake was negligible.

osmolarity. Expression is increased over 100-fold by in-creasing the osmolarity of the medium. Induction is rapidand is independent of the particular osmolyte used. The

lating high intracellular concentrations of compatible solutes molecular mechanism by which changes in external osmolar-such as proline or betaine which can serve both functions ity are sensed is obscure. It has been suggested that expres-(19, 20, 28). Betaine is known to play an important sion of the kdp operon is regulated in response to turgorosmoprotective role in several bacterial species (14, 18) and pressure rather than to external osmolarity per se (17). Ourto be accumulated to high concentration by E. coli in results strongly support this, providing evidence that turgorresponse to osmotic stress (23), although little is known pressure is responsible for proU induction. First, whileabout the mechanisms of betaine accumulation. In a previ- many solutes induce proU expression, glycerol has no effect.ous paper we have shown that the proP gene of S. Glycerol is known to permeate freely across the membranetyphimurium encodes a betaine transport system (1). How- and therefore, unlike the other solutes used, would elicit noever, in proP strains, there is a considerable betaine uptake effect on turgor pressure. Second, betaine and proline, butcomponent which is independent ofproP function. We show not other amino acids, substantially reduce expression ofhere that this additional transport component is dependent proU at high osmotic pressure. This is almost certainly anupon the proU gene. Mutations in proU were originally indirect effect. Thus, at high osmolarity these amino acidsidentified as conferring resistnce to the proline analog AC at are accumulated to restore turgor pressure. If proU expres-high osmotic pressure, and the gene was inferred to encode sion is mediated by turgor pressure, rather than by osmotican osmotically induced proline uptake system, PP-Ill (6). pressure per se, then such accumulation of betaine andHowever, we were completely unable to detect significant proline would be expected to reduce expression; this isrates of proline uptake through PP-III. On the other hand, precisely what is observed. Third, after a shift to highproU mutations reduced betaine transport rates consider- osmolarity proU expression initially increases rapidly, andably. Thus, proU encodes a transport system with betaine as the rate of synthesis is subsequently reduced as a steadyits primary substrate. Presumably, the transport system state is approached. This again is explained if turgor pres-lacks absolute specificity and can transport proline at low sure is the regulatory signal and after osmotic shift turgorrates (although insignificant compared with transport via pressure is partially, but never fully, restored (possibly byPP-I and PP-II), which accounts for the proU-dependent K+ uptake [8]). Finally, the induction profile for proUgrowth stimulation of proline auxotrophs reported by expression shows that, up to a certain threshold concentra-Csonka (6). It should be noted that we have not formally tion of external solute, proU expression is not induced.excluded the possibility that the proU gene is actually a While a variety of solutes can serve as osmoprotectants atpositive regulator of the betaine transport system, rather intermediate osmolarities, betaine is known to be the pre-than encoding the transport system per se. However, this ferred compatible solute at high osmolarity (28). It thereforeseems unlikely for two reasons. First, expression of a seems likely that, at these relatively low osmolarities, theregulatory gene would not be expected to be highly regu- cell is able to adjust turgor pressure by alternative meanslated, and second, operon fusions indicate a high level of such as increased K+ uptake (8) or proline synthesis (5).proU expression which is atypical of regulatory genes. In Above a critical value, turgor pressure can no longer beaddition, we have recently identified the proU protein, maintained by these means and proU is induced. The mostwhich is produced in very large amounts after osmotic reasonable mechanism by which changes in turgor pressureinduction (unpublished data). might be sensed is that structural changes resulting fromThe kinetics of proU-dependent betaine transport show it membrane shrinkage cause a conformational change in a

to be a high-affinity transport system (Km = 1.3 ,uM), membrane-bound regulatory protein (12, 17).particularly when compared with uptake via the ProP system proU-dependent betaine uptake is regulated at two levels.

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OSMOREGULATION OF GENE EXPRESSION 1231

Not only is proU transcription increased by increasingosmolarity, but even when induced, function of the transportsystem is only detected at high osmotic pressure. Regulationby osmotic pressure of both transcription and function isalso found for the proP betaine-proline transport system (1).Presumably the transport proteins only adopt an activeconformation at high osmolarity, again suggesting a ratherspecific regulatory role for changes in membrane conforma-tion.

It is of interest to ascertain the number of genes which areunder osmotic control and whether a single global regulatorymechanism is in operation. Besides proU, only the ompFand ompC genes and the kdp potassium transport operonhave previously been shown to be osmotically regulated(10-12, 17). To identify any other genes which might besimilarly regulated, we isolated six independent lacZ fusionsto the S. typhimurium chromosome whose expression wasosmoregulated. Each of these fusions was found to be in theproU gene. It must be considered that the screen adoptedhere would not detect genes which, unlike proU, are ex-pressed above a certain level even at low osmolarity (e.g.,ompC) or genes whose function is essential to the cell. Also,fusions to kdp would not have been detected since, in themedium used, K+ concentrations were such that kdp expres-sion is induced even at relatively low external osmolarity.However, despite these reservations it seems likely that onlya very limited number of genes ate regulated in response toosmotic pressure. This is in agreement with the observationthat the relative abundance of only a very few cellularproteins changes in response to increased osmolarity (4;unpublished data).Only one genetic locus has been identified which plays a

role in the osmoregulation of gene expression, the adjacentompR and envZ genes which are involved in the osmoregula-tion of ompF and ompC porin expression (10-12). Interest-ingly, we have recently found that ompR also plays a role inthe expression of genes which are not osmoregulated(Gibson and Higgins, submitted). The characteristics ofproU and porin expression are quite different. Thus, it is theratio of the two porins that is affected by medium osmolarity,and in addition, many other environmental factors can alsoinfluence their expression. Expression of proU, on the otherhand, seems to respond solely to turgor pressure. Expres-sion ofproU is effectively zero at low osmolarity, only beinginduced above a certain threshold level. The kdp operonseems to be regulated in a manner similar to proU (17). Wehave shown here that ompR and envZ mutations do notaffect the expression or osmoregulation of proU. Thus, theremust be at least two separate mechanisms by which tran-scriptional regulation in response to osmotic pressure isachieved.

ACKNOWLEDGMENTSWe thank Hugh Ainsworth for assistance with determining the map

location of the proU gene. We are grateful to Kelly Hughes and JohnRoth for providing us with phage Mu dl-8 before publication.

Financial support was provided by a grant from the MedicalResearch Council to C.F.H. and I.R.B. C.F.H. is a Lister InstituteResearch Fellow.

ADDENDUM

Since this manuscript was submitted a paper has beenpublished (V. J. Dunlop and L. N. Csonka, J. Bacteriol.163:296-304, 1985) which also demonstrates that osmoticinduction of proU expression is transcriptional.

LITERATURE CITED

1. Cairney, J., I. R. Booth, and C. F. Higgins. 1985. Salmonellatyphimurium proP gene encodes a transport system for theosmoprotectant betaine. J. Bacteriol. 164:1218-1223.

2. Cairney, J., C. F. Higgins, and I. R. Booth. 1984. Proline uptakethrough the major tranport system of Salmonella typhimurium iscoupled to sodium ions. J. Bacteriol. 160:22-27.

3. Casadaban, M. J., and S. N. Cohen. 1979. Lactose genes fusedto exogenous promoters in one step using a Mu-lac bacterio-phage: in vivo probe for transcriptional control sequences. Proc.Natl. Acad. Sci. USA 76:4530-4533.

4. Clark, D., and J. Parker. 1984. Proteins induced by highosmotic pressure in Escherichia coli. FEMS Microbiol. Lett.25:81-83.

5. Csonka, L. N. 1981. Proline over-production results in enhancedosmotolerance in Salmonella typhimurium. Mol. Gen. Genet.182:82-86.

6. Csonka, L. N. 1982. A third L-proline permease in Salmonellatyphimurium which functions in media of elevated osmoticstrength. J. Bacteriol. 151:1433-1443.

7. Csonka, L. N., M. M. Howe, J. L. Ingraham, L. S. Pierson, andC. C. Turnbough. 1981. Infection of Salmonella typhimuriumwith coliphage Mu dl(Apr lac): construction of pyr::lac genefusions. J. Bacteriol. 145:299-305.

8. Epstein, W., and S. G. Schultz. 1965. Cation transport in Esch-erichia coli. Regulation of cation content. J. Gen. Physiol.149:221-234.

9. Gibson, M. M., M. Price, and C. F. Higgins. 1984. Geneticcharacterization and molecular cloning of the tripeptidepermease (tpp) genes of Salmonella typhimurium. J. Bacteriol.160:122-130.

10. Hall, M. N., and T. J. Silhavy. 1981. The ompB locus and theregulation of the major outer membrane porin proteins ofEscherichia coli K12. J. Mol. Biol. 146:23-43.

11. Hall, M. N., and T. J. Silhavy. 1981. Genetic analysis of theompB locus in Escherichia coli K12. J. Mol. Biol. 151:1-15.

12. Hall, M. N., and T. J. Silhavy. 1981. Genetic analysis of themajor outer membrane protein of Escherichia coli. Annu. Rev.Genet. 15:91-142.

13. Hughes, K. T., and J. R. Roth. 1984. Conditionally transposi-tion-defective derivative of Mu dl(Ap lac). J. Bacteriol.159:130-137.

14. Imhoff, J. F., and F. Rodriguez-Valera. 1984. Betaine is themain compatible solute of halophilic eubacteria. J. Bacteriol.160:478-479.

15. Kennedy, E. P. 1982. Osmotic regulation and the biosynthesis ofmembrane-derived oligosaccharides in Escherichia coli. Proc.Natl. Acad. Sci. USA 79:1092-1095.

16. Kleckner, N., R. Chen, and B.-K. Tye. 1975. Mutagenesis byinsertion of a drug-resistance element carrying an invertedrepetition. J. Mol. Biol. 97:561-575.

17. Laimins, L. A., D. B. Rhoads, and W. Epstein. 1981. Osmoticcontrol of kdp operon expression in Escherichia ccli. Proc.Natl. Acad. Sci. USA 78:464-468.

18. Le Rudulier, D., and L. Bouillard. 1983. Glycine betaine, anosmotic effector in Klebsiella pneumoniae and other membersof the Enterobacteriaceae. Appl. Environ. Microbiol. 46:152-159.

19. Le Rudulier, D., A. R. Strom, A. M. Dandekar, L. T. Smith,and R. C. Valentine. 1984. Molecular biology of osmoregula-tion. Science 224:1064-1068.

20. Le Rudulier, D., and R. C. Valentine. 1982. Genetic engineeringin agriculture: osmoregulation. Trends Biochem. Sci. 7:431-433.

21. Menzel, R., and J. Roth. 1980. Identification and mapping of asecond proline permease in Salmonella typhimurium. J. Bacte-riol. 141:1064-1070.

22. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

23. Perroud, B., and D. Le Rudulier. 1985. Glycine betaine trans-port in Escherichia coli: osmotic modulation. J. Bacteriol. 161:

VOL. 164, 1985

on Septem

ber 27, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

1232 CAIRNEY ET AL.

393-401.24. Ratzkin, B., and J. Roth. 1978. Cluster of genes controlling

proline degradation in Salmonella typhimurium. J. Bacteriol.133:744-754.

25. Roth, J. R. 1970. Genetic techniques in studies of bacterialmetabolism. Methods Enzymol. 17A:3-35.

26. Stalmach, M. E., S. Gruthe, and J. M. Wood. 1983. Two proline

porters in Escherichia coli K-12. J. Bacteriol. 156:481-486.27. van Alphen, W., and B. Lugtenberg. 1977. Influence of osmolar-

ity and the growth medium on the outer membrane proteinpattern of Escherichia coli. J; Bacteriol. 131:623-630.

28. Yancey, P. H., M. E. Clark; S,. C. Hand, R. D. Bowlu, andG. N. Somero. 1982. Living with water stress: evolution ofosmolyte systems. Science 217:1214-1222.

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