Induction of Lactose Transport in Escherichia coli During the Absence of Phospholipid Synthesis

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1 JOURNAL OF BACTERIOLOGY, Aug. 1975, p Copyright 1975 Americn Society for Microbiology Vol. 123, No. 2 Printed in U.S.A. Induction of Lctose Trnsport in Escherichi coli During the Absence of Phospholipid Synthesis LAURIE J. WEISBERG, JOHN E. CRONAN, JR.,* AND WILLIAM D. NUNN Deprtment of Moleculr Biophysics nd Biochemistry, Yle University School of Medicine, New Hven, Connecticut 651 Received for publiction 28 April 1975 Induction of lctose trnsport nd of o3-glctosidse synthesis ws exmined in two Escherichi coli strins tht require exogenous glycerol for phospholipid synthesis nd growth. No preferentil inhibition of lctose trnsport induction ws observed when phospholipid synthesis ws restricted to 5 to 1% of the norml rte. We conclude tht the lctose trnsport system does not require concurrent phospholipid synthesis for its functionl ssembly. Severl yers go, Hsu nd Fox (6) reported tht inhibition of phospholipid synthesis in Escherichi coli (by strvtion of glycerol uxotroph for glycerol) resulted in drstic decrese in the inducibility of lctose trnsport reltive to tht of 3-glctosidse. These uthors interpreted their results s indicting tht induction of lctose trnsport requires de novo phospholipid synthesis. Similr experiments using glycerol uxotrophs of Bcillus subtilis nd Stphylococcus ureus, however, showed tht neither citrte trnsport in B. subtilis (11) nor lctose trnsport in S. ureus (7) required concurrent phospholipid synthesis for norml induction. In ddition, recent work (8. 9) hs shown tht induction of lctose trnsport in E. coli does not require concurrent unsturted ftty cid synthesis s ws first believed (5). The results of Hsu nd Fox (6) re the only dt supporting the notion tht trnsport system induction requires concurrent phospholipid synthesis. The bove rguments nd the existence of severl experimentl flws in the work of Hsu nd Fox (6), led us to reexmine the dependence of lctose trnsport induction on phospholipid synthesis. In contrst to the previous results, we find tht lck of phospholipid synthesis does not result in preferentil inhibition of lctose trnsport induction. MATERIALS AND METHODS Bcteri, medi, nd growth conditions. The two bcteril strins used re derivtives of E. coli K-12. Both strins re derived from strin 8 of Lin (see reference 1) nd thus re unble to ctbolize either glycerol or sn-glycerol-3-phosphte (G3P). Both strins re dependent upon exogenously suppled glycerol (or G3P) for phospholipid synthesis nd growth. Strin BB26-36 contins muttion in the plsb locus nd hs defective membrnous G3P cyltrnsferse (1, 4). The cyltrnsferse is functionl but hs n pprent Km for G3P tht is lest 1 times higher thn the Km of the norml enzyme. Strin BB2-14 hs muttion in the gpsa gene which codes for the biosynthetic G3P dehydrogense (1, 3), nd thus hs no detectble dehydrogense ctivity. The bcteri were incubted in gyrtory wter bth shkers t 37 C. Cultures were grown in stndrd medium of low phosphte [LP medium: 1 mm KPO4,.1 M tris(hydroxymethyl)minomethne-chloride, 1 mm KCl, 15 mm (NH4)2SO4, 1 mm MgCl2, ph 7.2] supplemented with.4% sodium succinte nd.5% vitmin-free csein hydrolyste. When pproprite, glycerol ws dded to finl concentrtion of.2%. Induction of the lctose operon. The inducer used ws isopropyl-3-d-thioglctoside t finl concentrtion of 1 mm. Lctose trnsport ssy. The ssy mixture (1.2 ml totl volume) consisted of.5-ml smple of bcteril culture,.2 ml of 13 mm o-nitrophenyl O-D-glctoside (ONPG) nd.5 ml of LP medium contining chlormphenicol (finl concentrtion of chlormphenicol ws 42 gg/ml). A prllel ssy tube contined ll of these components plus 1-thio-3-Ddiglctoside ( specific inhibitor of lctose trnsport) t finl concentrtion of 7.6 mm in the ssy mix. The ssy tubes were incubted t 37 C for 17 min, nd the rection ws quenched by the ddition of.5 ml of 1 M N2CO3. The cells were removed by centrifugtion nd the relese of o-nitrophenol ws quntitted by mesuring bsorbnce t 42 nm in Colemn Jr. spectrophotometer. The reding for the ssy tube contining 1-thio-O-D-diglctoside ws subtrcted from the reding for the experimentl (no 1-thio-f3-D-diglctoside) tube thus giving vlue for the lctose operon-medited trnsport of ONPG. This is essentilly the sme ssy previously used in this lbortory (8). Assy of totl j3-glctosidse. The mixture used for ssy of this enzyme (totl volume 1.2 ml) 492 Downloded from on Jnury 9, 219 by guest

2 VOL. 123, 1975 consisted of.1-ml smple of bcteril culture,.4 ml of LP medium,.2 ml of 13 mm ONPG,.5 ml of LP medium contining chlormphenicol (finl concentrtion 42 ug/ml), 1 drop of 1% sodium dodecyl sulfte, nd 2 drops of chloroform. The tubes were vigorously mixed on Vortex mixer nd the ssy ws run for 17 min in prllel with the trnsport ssy. The rection mixtures were then quenched with crbonte nd red s given bove. The detergent nd chloroform were used to disrupt the cellulr permebility brrier (8) Ṙte of phospholipid synthesis. After 12 min of the 17-min induction period for ech time point, 1-ml smple of induced cells ws removed from ech induction flsk, plced in tube contining 4 gci 32Pi (4 MCi/umol) nd shken in the sme wter bth. After 5 min of incubtion, the lbeling ws hlted by the ddition of 6 ml of methnol-chloroform (2:1). Lipids were extrcted nd quntitted s previously described (8) with the modifiction tht the originl extrction mixture ws divided into two portions nd the procedure ws crried out in duplicte. Removl of glycerol. Cultures of bout 2 x 18 cells/ml growing exponentilly in the stndrd medium plus glycerol were filtered on.45 gm membrne filter (Millipore Corp.), wshed 1 times with few milliliters of stndrd medium, nd resuspended in volume of stndrd medium similr to the originl volume. The culture ws divided into two portions, one of which received glycerol to finl concentrtion of.2%. Cell growth ws determined t 54 nm in Klett-Summerson colorimeter. One Klett unit equls bout 5 x 16 cells/ml. Rdioctive phosphte ws from New Englnd Nucler. The csein hydrolyste ws from Nutritionl Biochemicls Corp. The,B-glctoside regents nd chlormphenicol were from Sigm. RESULTS We found the results of Hsu nd Fox (6) difficult to interpret for two mjor resons. First, these workers grew their cultures on mixed crbon source of glycerol nd succinte. When the glycerol uxotroph ws deprived of glycerol, therefore, the culture encountered crbon source shiftdown in ddition to n inhibition of phospholipid synthesis. Since this type of crbon source shiftdown might be expected to ffect induction of the lctose operon (2), it is difficult to ttribute the dt obtined in these experiments solely to n inhibition of phospholipid synthesis. Second, Hsu nd Fox ssyed lctose trnsport by mesuring intrcellulr ccumultion of rdioctive glctoside. They presented no controls for pssive lekge of the ccumulted glctoside. An pprent lck of trnsport, then, could be due to the msking of trnsport by pssive lekge. We hve voided these difficulties in our experiments. Crbon source shiftdown ws LACTOSE TRANSPORT INDUCTION 493 voided by using glycerol uxotrophs tht were lso defective in glycerol ctbolism. Complictions due to efflux of trnsported glctosides were eliminted by using the trnsport (nd subsequent hydrolysis) of ONPG to ssy lctose trnsport. The ONPG trnsport ssy is unffected by efflux (8). Two different types of glycerol (or G3P) uxotrophs were used in these studies. Strin BB26-36 hs defect in the plsb gene which results in sn-glycerol-3-phosphte cyltrnsferse with Km for G3P tht is 1-fold higher thn norml (1, 4). This strin is therefore defective in utiliztion of endogenous G3P nd requires exogenous supplementtion with glycerol or G3P to llow norml phospholipid synthesis nd growth. After glycerol deprivl, strin BB26-36 continues to grow for bout one genertion, lthough phospholipid synthesis is immeditely reduced by more thn 9% (Fig. 1). Bell (1) hs shown tht nucleic cid synthesis continues lmost normlly for bout one-hlf genertion fter the onset of glycerol strvtion, wheres protein synthesis continues t only 5 to 6% of the norml rte for genertion fter strvtion. In light of these chrcteristics of the strin, induction of lctose trnsport ws ssyed in the period fter glycerol deprivtion during which cellulr physiology (excepting phospholipid synthesis) ppers firly norml. We find tht induction of both lctose trnsport nd f-glctosidse is inhibited by bout 5% in glycerol strved cultures of strin BB Thus, no preferentil inhibition of lctose trnsport induction ws found lthough phospholipid synthesis ws inhibited by greter thn 9%. The prtil inhibition of the induction of both lctose operon functions is probbly due to the prtil inhibition of bulk protein synthesis observed by Bell (1) nd by Nunn nd Cronn, (mnuscript submitted for publiction). Hsu nd Fox (6) used strin of E. coli defective in the biosynthetic G3P dehydrogense nd hence deficient in G3P synthesis. The biochemicl defect in the strin used by these workers differs from the lesion in strin BB To eliminte this disprity, we repeted our experiments with strin BB2-14 which like the Hsu nd Fox strin hs defect in the gpsa locus, the structurl gene for the G3P dehydrogense (1, 3). The induction of lctose trnsport nd fl-glctosidse in strin BB2-14 re only bout 5% decresed in glycerol-strved cells, lthough phospholipid synthesis is more thn 94% inhibited (Fig. 2). Thus, in strin BB2-14, s well s in strin BB26-36, no Downloded from on Jnury 9, 219 by guest

3 494 WEISBERG, CRONAW, AND NUNN Ln c =, technique for the lysis of E. coli for the ssy of' ) glctosidse. This technique ws shown to give higher vlues of enzyme ctivity (probbly B + Glycerol owing to enzyme stbiliztion) thn do most of' 1 75 O 5 ' tn -T ) n C. C~ 75 c 75 6z~ ) cw 25F 6 4 2k FA T I T r_preferentil inhibition of lctose trnsport in- BB26-36( p/s8-) duction is cused by drstic inhibition of phospholipid synthesis. The results of the experiments given in Fig. 1 nd 2 re tbulted in + glycerol Tble 1, nd re representtive of lrge e5t number of experiments, none of which show ny specific inhibition of lctose trnsport induction during glycerol strvtion. In ll these experiments, the levels of' inhibition of phospholipid j.s -glycerol synthesis were equl to or greter thn the inhibition observed by Hsu nd Fox (6) Minutes fter resuspension Since the completion of this work, pper by Putnm nd Koch (1) reported n improved the techniques commonly used in the literture. To test if our lysis method lso yields less thn the mximl 3-glctoside ctivity, we hve 13-goIct ssyed the 3-glctosidse ctivities of induced cultures of strins BB26-36 nd BB2-14 (both glycerol strved nd unstrved). We consistently found 1.9 to 2.-fold more ctivity in ll cultures using the method of Putmn nd Koch l"b--o (1). Therefore, ech of the 3-glctosidse Trnsport vlues reported in Fig. 1 nd 2 should be multiplied by this fctor. - G y c e r o \ *_ Trnsport A.. _^ -l.. lycerol -glycerol J. BACTERIOL. DISCUSSION We hve found no preferentil inhibition of lctose trnsport induction when phospholipid synthesis is decresed to only bout 5% of' the norml rte. Owing to these results, we see no reson to believe tht phospholipid synthesis is needed for the ssembly of functionl lctose trnsport system. It should be noted tht the inhibition of trnsport induction reported by FIG. 1. Rtes of induction of lctose trnsport nd of f3-glctosidse in the presence or bsence of glycerol. Strin BB26-36, plsb- strin, ws grown, then deprived of glycerol by filtrtion nd resuspended in either the presence or bsence of glycerol s described in Mterils nd Methods. At the times indicted, smples were removed, induced with isopropyl-f-d-thioglctoside nd incubted for 17- min induction period fter which they were ssyed for lctose trnsport nd d3-glctosidse ctivity. After 12 min of the 17-min incubtion period, smples were removed to flsk contining 32Pi nd incubted for 5 min before lipid extrction. Experimentl detils re given in Mterils nd Methods. (A) shows cell growth 1 Klett unit equls bout 5 x 16 cells/ml) plus nd minus glycerol; (B) nd (C) show induction of lctose operon function in cultures plus (B) or minus (C) glycerol. (D) shows the rtes of phospholipid synthesis in the presence or bsence of glycerol. Downloded from on Jnury 9, 219 by guest

4 VOL. 123, 1975 to. 4- (D -d z U) o U) vx cs CD - C - lz c#a w U) u o 1-. CL ii lO I I I I B. + Glycerol 3-golct. Trnsport C. -Glycerol D. * \,8-golct. Trnsport,lol -glycerol.-. o T i I I *. I Minutes fter resuspension FIG. 2. Rtes of induction of lctose trnsport nd of fl-glctosidse in the presence or bsence of glycerol. The experiments nd designtions re identicl to those of Fig. 1 except tht strin BB2-14, gpsa- strin, ws used. LACTOSE TRANSPORT INDUCTION 495 TABLE 1. Function ssyed Effects of glycerol deprivtion Inhibition (%) Strin BEB26-36 BB2-14 pisb - gpsa - Phospholipid synthesis 91.2 i ±.4,B-Glctosidse induction 44.6 ± ± 7.8 Lctose trnsport induction 53.4 i i 7.2 Rtio f,-glctosidse inhibition trnsport inhibition Men of inhibitions clculted from the dt for ech time point in Fig 1 nd 2 (plus glycerol vlue = 1%) + stndrd devition from the men. Hsu nd Fox (6) ws found only upon prolonged glycerol strvtion. Since they lso found tht the synthetic rtes of ll mcromolecules tested were bnormlly low fter prolonged strvtion, their results re most difficult to ttribute solely to inhibition of phospholipid synthesis. We lso exmined cultures fter prolonged glycerol strvtion (dt not shown) nd found tht induction of both lctose trnsport nd,b-glctosidse re both mrkedly inhibited by such tretment. Our present nd previous (8) results together with those of Mindich (7) nd Willecke nd Mindich (11) nd of Overth et l. (9) indicte tht bcteri cn induce functionl trnsport systems even in the bsence of de novo phospholipid synthesis. This result is true for ll trnsport systems thus fr exmined. ACKNOWLEDGMENTS This work ws supported by Public Helth Service grnt AI-1186 from the Ntionl Institute of Allergy nd Infectious Diseses nd Ntionl Science Foundtion grnt GB J. Cronn is supported by Public Helth Service Reserch Creer Development Awrd GM-7411 from the Ntionl Institute of Generl Medicl Sciences. W. Nunn is supported by Public Helth Service Postdoctorl Fellowship 1 F2 AI 55,327 from the Ntionl Institute of Allergy nd Infectious Diseses. LITERATURE CITED 1. Bell, R. M Mutnts of Escherichi coli defective in membrne phospholipid synthesis: mcromoleculr synthesis in n sn-glycerol 3-phosphte cyltrnsferse Km' mutnt. J. Bcteriol. 117: Contesse, G., M. Crepin, F. Gros, A. Ullmnn, nd J. Monod On the mechnism of ctbolite repression, p In J. Beckwith nd D. Zipser (ed.), The lctose operon. Cold Spring Hrbor Lbortory, New York. 3. Cronn, J. E., Jr., nd R. M. Bell Mutnts of Escherichi coli defective in membrne phospholipid synthesis: mpping of the structurl gene for L-glycerol 3-phosphte dehydrogense. J. Bcteriol. 118: Downloded from on Jnury 9, 219 by guest

5 496 WEISBERG, CRONAN, AND NUNN 4. Cronn, J. E., Jr., nd R. M. Bell Mutnts of Escherichi coli defective in membrne phospholipid synthesis: mpping of sn-glycerol 3-phosphte cyltrnsferse K,, mutnts. J. Bcteriol. 12: Fox, C. F A lipid requirement for induction of lctose trnsport in Escherichi coli. Proc. Ntl. Acd. Sci. U.S.A. 63: Hsu. C. C., nd C. F. Fox Induction of the lctose trnsport system in lipid-synthesis-defective mutnt of Escherichi coli. J. Bcteriol. 13: Mindich, L Induction of Stphylococcus ureus lctose permese in the bsence of glycerolipid synthesis. Proc. Ntl. Acd. Sci. U. S. A. 68: Nunn, W. D., nd J. E. Cronn, Jr Unsturted J. BACTERIOL. ftty cid synthesis is not required for induction of lctose trnsport in Escherichi coli. J. Biol. Chem. 249: Overth, P." F. F. Hill, nd I. Lmnek-Hirsch Biogenesis of E. coli membrne: evidence for rndomiztion of lipid phse. Nture New Biol. 234: Putnm, S. L., nd A. L. Koch Complictions in the simplest cellulr enzyme ssy: lysis of Escherichi coli for the ssy of W-glctosidse. Anl. Biochem. 63: Willecke, K., nd L. Mindich Induction of citrte trnsport in Bcillus subtilis during the bsence of phospholipid synthesis. J. Bcteriol. 16: Downloded from on Jnury 9, 219 by guest