FEMS M!:robiologyLetters 69 (19qO) 19-26 l%biished by Elsevier

19

FEMSLE 03975

Acid shock proteins of Escherichia coli M a r t i n e H e y d e and R a y m o n d Portalier Lab-vratowe tic Mtcrobtofogie et G~n~tJque Moldculatre ( U M R C?~'RS ILM). B,~timenl 405, UnWerwt~Claude Bernard Ll.'on f. F-h~622 Villeurt~mne Cedex France

Received 12 December 1989 Accepted 8 January 1990 Key words: Escherichia co/t; Heat shock proteins: pH stress; RpoH sigma factor; Electrophoresis, bi-dimensional

1. S U M M A R Y Synthesis of total cellular proteins of E w h e richia cob was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins indOced by such an external pH shift down were identified by mend- and bi-dimensional electrophoresis. 30 to 45 rain after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DunK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and

Corre.~pondence to: M. II¢$de, Lab0rotoire de Microbiologieet G~n6'tique Mol6culaire. B,~ttiment 405. Universit6 Claude Bernard Lyon 1. 69622 VilleurhanneCedex. France. Abbreviations: gpoH, a special sigma subunits (032) of RNA polymeras¢; ASP, Acid Shock Protein; HSP, Heat Shock Protein; HOP, High Osmotic pressure PTotein; LB, Luria Broth, phi, intraccUular pH ; pile. ¢xtraeel|ular pHz v/v. by vol.: w/v, mass/vol. Enzymes: RNA polymetase (EC 2.73.fi1; dihydrolipoamide acelyltransferase (EC 2.3.1.121; phosphorylase B (EC 2A,I,I); carbonic aahydrase (EC 4.2.1.1.L

C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP), The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.

2, I N T R O D U C T I O N Escherwhia edit, like many organisms must cope with a wide variety of environmental stresses. Multigene systems [1] provide a way for E, coli to respond and adapt to stimuli such as hca~. [2], pH [3A}, anaerobiosis [5,6], aerobiosis [7}, D N A damage [8], osmolarity [9,10] and phosphate limitation NIl. The heat shock response, which has been described in a wide range of organisms is the most well documented stress system. In E. colt, the heat shock response is characterized by a rapid increase in the synthesis of 17 proteins called the heat shock proteins {HSP) [121. These proteins define a regulon which is positively controlled by a new sigma factor, the RpoH protein or sigma 32 [13,14]: There are many inducers of the heat shock response in E. coli. The most potent is a shift up in temperature but ethanol, puromycin, viral infection, nalidixic acid mimick heat itself (for re-

0378-1097/90/$03.50 © 1990 Federation of European MlclobiologicalSocieties

20 view see [1~,). But, it is not general stress response since related SOS and oxidative regulons can be independently induced [1]. However, the evolutionary conservation of heat shock proteins suggests they might be involved in cellular homeostasis. The mechanisms used by E. colt to adapt to variations of intracelhilar (pHi) or extraeelhilar (pHo) pH are only poorly understood. After a shift up to pHo from 6.6 to 8.8, E. col~ displays a capacity for pH homeostasis and can maintain its pHi to 7.6 t15]. To identify the mechanism which might contribute to pH homeostasis and adaptation, proteins whose concentrations are modified by pH shifts were researched by bi-dimensional electrophoresis analysis. In this paper, we show that an acid shift partially induces the heat shock response as well as a set of seven polypeptides never described previously to be induced by any stress. The role of the regulatory RpoH protein in this induction pattern is discussed.

3. MATERIALS AND METHODS

3.1. Bacterial strains All bacterial strains used in this study are derivatives of E. colt K-12. Strain K165 F - lacZ53 (Am) phoA5(Am) supC47 supC91(Ts) irp48(Am)

relA1 rpsL150 maiTO6(Am) rpoIt165(Am) spoT1 toaA22 ompF627 phoU35 bglCop16 was previously described [16]. Strains MA3337 (maIT::TnlO rpoH ~ ) and MA3334 (malT: :Tn 10 rpoPl165) were derivatives of strain Kt65,

3.2. Media and growlh conditions Cultures in rich Mops medium supplemented with 22 mM glucose, 5 vitamins, 4 bases and 20 amino acids, as described by Wanner [17], were incubated at 30°C under aeration until they reached ~ density of 4 x 108 cells per ml. pH of Mops medium, initially adjusted to pH 6.9 did not vary significantly during growth, pH shifts, from pH 0,9 to lower pHs were performed by adding aliquots of 1 N HCI in cell suspensions incubated at 30"C. pH and osmolarity of all suspensions were measured before and after every experiment.

After a pH shift-down from pH 0.9 to pH 5, 4, 3 or 2 or a temperature shift-up from 30 to 50 ° C, cell viability was estimated by spreading appropriate dilutions of the treated suspensions on Luria Broth plates [18] and counting survivors. Ten minutes after a pH shift (pH 6.9 to pH 4.3) or a temperature shift (30 to 50 ° C), cellular viability decreased to about ~0%, After more drastic acid shifts from pH 6,9 to pH 3 and pH 2, cdlular viability was lower than 1O'~ whereas no significant modification of viability was observed after a pH shift-down from pH 6.9 to pH 5. On the other hand~ no growth was observed when E. colt cells were inoculated in LB medium adjusZed at pH lower than 5,

3.3 Synthesis of total celhdar proteins Cells were grown at 30°C in rich Mops medium. After a pH or a temperature shift, they were pulse-labelled for 1 min 1 ~Ci/ml of mixture of 15 L-IJHlarnlno acids, then treated for 15 min with an equal volume of cold 10% trichloracetic acid. Samples were filtered through Sartorius 0.45 v.m filters and cells were washed with 20 ml of cold 5% trichloraeetie acid and 2.5 ml ethanol before drying. Acid-insoluble radioactivity retained on filters was determined with a liquid scintillation counter (LSlg00-Beckman) using Ready Solv H P / b scintillation solution (Beckman, France).

3,4. Sample preparation and gel electrophoresis Total cell extracts who~e protein composition was analyzed by SDS-PAGE were prepared by washinS cells with 10 mM Tris buffer pl! 7 containing 5 mM MgCI~ and resuspending them in cracking buffer [19]. Electrophoresis was performed as described by Laemmli [19]. Total cell extracls analyzed by two-dimensional polyaerylamide gel ¢lectrophoresis [20] were prepared aeco.,'ding ~,J Cortay et a!. [21]. Separation h~ the first dimension was achieved by isoel~tric focusing to equilibrium (11000 V a i l , houO in pH 5-7 of pH 3-10 (by col iv/v)) ampholines. In the second dimension, gels were run at 800 Volt- hour in 12.5~ (mass/col ( w / v / ) ) polyacrylamide and 0.1~ (w/v) SDS.

21

Before t'luorography and autoradiography, gels were incubated for 30 rain in 7% ( v / v ) acetic acid. l~sS]-labelled proteins were detected by fluorogra. phy after incubation for 20 rain in Amplify (Amersham); gels were dried under vacuum and exposed at - 8 0 ° C to Fuji RX100 films,

A +

E

u

3.5. Malerials [35S]methionine SJ204 (1070 Ci/mmol), L[~H]amino acids mixture TRK440 0 3 to 109 C i / m m o l ) and t4C-labdled proteins for molecular weight standards were obtained from Amersham International (U.K.), Fuji RX100 films were purchased from C G R Thomson (France).

o

"a m

+++.+,+++.+. ~

:~

.'..

+

+~+

ID

B

e_ ,a o

4. RESULTS

4.1. Kinetics of total protein ~ynthesis following an acid pH shift Shifting cells of strain MA3337 from pH 6.9 to pH 4,3 led to the same decrease in cellular viability than that observed after a temperature shift-up from 30 to 5 0 ° C (see MATEIR1ALSAND MI!THODS). To analyze the variation in overall protein synthesis induced by lowering extracellular plt, cells were pulse-labelled at various times before (0 time) and a~'ter pH shifts, Data illustrated of Fig. 1A show that within 2 min after a shift from pH 6.9 to pH 4.3, protein synthesis decreased about 3-fold, then reached a new steady-state value equal to about 70% of the control value. Th~ pattern o.~ protein synthesE w~s ve,~' d;,fferem from the one observed after a temperature shift (Fig. 1B). in agreement with previously published resalts [22], we noticed thai after a temperature shift-up of strain MA3337 from 30 ~.o 5¢2°C, protein synthesis increased within 30 s, then reached a steady state level after 40 rain (Fig. 1B)+ When cells were exposed to a more drastic acid shift from pH 6.9 to pH 2.2, protein synthesis decreased abruptly within 2 rain and did not recover (Fig. 1A); in these conditions, cellular viability was lower than 10% (See ~#_~T~.t^t.S *..,qD.,,m~no~).

4.2. Specific proteins are induced by an acid shift Proteins specifically induced by an acid shift were identified, after labelling, by SDS-PAGE

Time (rain)

Fig. 1. influence of a pH or a temperature uhift on total protein synthesis. Strain MA3337 was grown in rich Mops medium as described MxTEglALSAND METHODS.At different times before or after a shift, 100 ~tl of ¢¢1t suspension were pulse-labelled for t wan with I ~Ci/ml of a mixt~r~ of L-t5 [3Hlamino acids. Acid insoluble radioactivitywas measured as described in saATI~RIALSAND METHODS.A: control (Ak pH shifts: pH 6.9 to pH 4.3 (e), pH 6.9 to pH 2.2 (a); B: temperature shift: 30 ° C to ~0 ° C (O). (Fig. 2). The protein pattern of total extracts prepared from cells labelled 3 rain after shift from pH 6.9 to pH 4.3 and during 15 rain was characterized by a very low content (Fig. 2 compare lanes a and c). in agreement with protein synthesis kinetics (Fig. IA). On the other hand, if similar samples were labelled during 45 rain, several polypeptides could be identified whose contents were significantly increased (Fig. 2 compare lanes d and e). Four of these polypeptides showed similarity of migration pattern with the 87 kDa, DnaK (74 kDa), GroEL (64 kDa) and 61 kDa heat shock proteins described by Yamamori [221 which were induced by a temperature shift in rpoH+ but not in rpoH- strains (Fig. 2 compare lane e with lanes a, b. f and g). These results were confirmed by bidimensional electrophoretie analysis (as see below). At least four other polypeptides with apparent molecular masses of 90 kDa, 85 kDa, ,$3 kDa and 27 kDa were specifically and reproduei-

22

a

b

c

de

f

! ~

B

kOa

bly increased by a pH shift but not by a temperature shift (Fig. 2 lanes d, e and b) or an osmolarity shift (data not shown). Similar results were obtained after pH shifts from pH 6.9 to pHs in the

•

OA$1£

ACIO I ~

~

~

I

I

I

64~ 61~ dip. -24

46

--IS

it

kDa

30~

-Sl. - 7| -CO

Fig, 2. Monedimensional SDS-PAGE patterns of total proteins after a pH or a temperalure shiR. Strains MA3337 (rpoH + ) (lanes a-e) and MA3334 (epoH155) (lanes f and g) were grown at 30°C in methionine-free rich Mops medium. Cells were labelled with 30 pCi/m] of [3~S]rnethionine during 15 (lanes a, b, c, f and g) or 45 rain (lanes d and e), before (controls, lanes a, d and f) or 3 rain after a tempezatufe shift (30 to 50°C, laae~ b and g) or a pH shift (pH 6,9 to 4.3, lanes c and c), Incorporation was stopped b3~ adding cold rnethionine (0,05 mM) and whole cell extracts ~ere analy~ed by SDS-PAGE (8-15% acrylamide 8radient). l0 s cpm samples were loaded on gels. After fluorography, films were expl,s~ for 24 h, Protein molecular masses were determined from the mtgJralion pattern of a t~C-pmtein standard mixture containing phospho~tasc B (92,5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa) and carbonic anhydrase (30 kDa). Closed cirelas (O) indicate polypeplide~ whose synthesis was induced by both pH and temperature shifts whereas arrow heads point out polypeptides whose synthesis was specifically induced by a pH shift,

-24

Fig. 3. Bidimensional eleetrophoretic pattern of proteins synthesized after an acid shift. Strain MA3337 was Down at 30°C in melhionine-ftee rich Mops medium. Before (controlA) and thr~ minutes after (B) a pH shift (pH 6.9 to pH 4.3), aliquots were labelled for 30 rain with ~ /tCi/rnl of [3SSlrnethionine. Radioactivity was chased with cold rnethionine and 1oral cell extracts, prepared as described in ~ T ~ ^ ~ AND U~rHODS were anal?/zed by two-dimensional electmphoresis, Samples t,quivalent to 2 x lO~ crop were loaded on gels. Aft= fluorography, films were exposed for 24 h. The position ,~f polypeptides whose synthesis was reprodacibly induced by a pH shift alter five independent labelling experiments is indicated by circles, squares and arrow heads. Spots marked with a bracket were not modified after a pH shift and were used as internal controls.

23 r a n g e o f 4 to 3.3 p H shock polypeptides, however, were n o t i n d u c e d by a n a r r o w e r shift from p H 6.9 to p H 5 ( d a t a n o t shown).

4.3. Identification of proteins induced by an acid

shi/t The protein p a t t e r n o f total cellular extracts labelled for 30 m i n after a shift f r o m p H 6.9 to p H 4.3 w a s analyzed by b i d i m e n s i o n a l electrophoresis a c c o r d i n g to O ' F a r r e l l [20] Fig. 3). At least sixteen p o l y p e p t i d e s with m o l e c u l a r m a s s e s higher t h a n 30 k D a were p r e s e n t in increased a m o u n t s o r were n e w l y synthesized after an acid shift (Fig. 3, s p o t s m a r k e d with arrow heads, circles a n d squares). S p o t s n u m b e r e d 2, 3, 6, 10, 13 a n d 17 (tagged with circles on Fig. 3 a n d Fig. 4A) displayed the s a m e m i g r a t i o n p a t t e r n as polypeptides also i n d u c e d by a t e m p e r a t u r e shift-up in a rpoH* strain (Fig. 4: c o m p a r e a u l o r a d i o g r a m s a a n d c; Table 1). Spot~

n u m b e r e d 2, 3, 6, a n d 10 c o r r e s p o n d s to heat shock proteins G r u E L , D n a K , H t p G a n d H t p M , respectively (Table 1) [23,24]. S p o t s 2, 3, 6, a n d 10 are also i n d u c e d by ultraviolet light a n d nalidixic acid, b u t i n d e p e n d e n t l y of the SOS r e g u l a t o ~ s y s t e m [25]. After a n acid p H shift o f lower r a n g e ( p H 6.9 to p H 5), two o t h e r s p o t s ( n u m b e r e d 1 a n d 7 on T a b l e 1) were induced, which m i g r a t e d as the heat shock p r o t e i n s G r p E a n d L y s U , respectively ( d a t a not shown). Spot 32 (squared o n Fig. 3 a n d Fig. 4 a, b a n d d) was intensified after p H a n d o s m o l a r i t y shifts (Table 1). It m i g r a t e d as o n e of the high o s m o t i c p r e s s u r e p r o t e i n s ( H O P ) described by Clarke [9], (Fig. 4 a a n d d, s q u a r e d spots). N o n e o f t h e H O P were i n d u c e d by heat shock. O n the o t h e r h a n d , 9 p o l y p e p t i d e s which were i n d u c e d by an acid shift (spots n u m b e r e d 61.62, 63, 66, 67, 70, 72, 73 = a J 74) (Figs. 3 a n d 4b arrow

Table l Summa~ data

¢[

;he protons induced by pH. lomperatur¢ or osmolarity shirts as illustrated in Figs 3 and 4

Nature o f proteins Alphanumeric name designation

Strain rpoH--inductionby • decreased increased pH temperature

1 2 3 4 5 6

B25.3 GrpE B56.5 GruEL B66.0 DanK C14.7 HpIE C15.4 G~oES ('62.5 HtpG

+ + ND ND +

7

I)60.5

Spot number

LysU

Strain rpoHI65--induction b?/ increa~d osmolarity

pH

+ 4 + f + J.

. .

. . ND ND -

-

_+

.

+ + + +

+ +_ + -

. +

l0 13 17 32

F84.1 HIpM

61

C70,0

+

-

62

C62.7

+

63

. .

.

temperature

osmolarity -

. .

.

-

-

.

. +

-

-

. -

+

-

+

-

-

-

-

÷

-

-

+

-

-

÷

-

-

66

+

-

-

+

-

67

+

-

-

÷

-

-

70

+

-

-

+

-

-

72

+

73

+

-

+

-

-

74

+

-

+

-

-

.

-

.

.

.

.

Spots were numbered as indicated in Figs. 3 and 4. Proteins were identified by the alphanumeric code of N¢idhardt [23| indicating Iheir position on two-dimensional eleclrophorcticgels. Only spots which were reproductibly induced by an acid shift through five independent experiments were indicated in this Table. pH, temperature and osmolafity shifts wore performed according to the legend o f F i g . 4 . - : no induction, :k or + : low or high induction, r~spectlv¢ly: ND: low molecular weight polypeptides not delected on the autoradiograms (Figs 3 and 4).

24 ~'~z~

.~_~e ~

.~_.~'~~ ~ L

J

~.-~.~ ~

~._

o~

.~

'~, ~' v

J

d~

-:

• ....... ~,

.,.o~

~,~ :~ t.

®

'0 .

,

i

o

,

"{~'.."~'~ ~ 0 "

~; ,m

~

,~ c

~, ~ ~'~

.

.~. .-

~ _ ~

.-o~ t,.-,

I= to .1~ ..~

~

~..~

.o

25 head symbols) were not induced by a temperature or an osmo[arity shift (Fig. 4 a, c, d; Table 1). Spots 61 and 62 m/grated as the dlhydrolipoamide acetyltransferase subunits (C70.0 and C62.7) and were described to be induced by a low temperature or an aerobiosis shift [7,26]. Spots 63, 66, 67, 70, 72, 73 and 74 did not migrate as polyF.~ptides previously described to be induced by low temperature, UV light, aerobiosis, anaerobiosis or pH stress. As they were specifically induced by an add shift we called them acid shock proteins (ASP).

4.4 Role of RpoH in the synthesis of the acid shock proteins As expected, mutant strain MA3334, which is deficient in RPOH, a sigma suburfit (o ~2) of RNA polymerase specifically involved in the initiation of transcription at the heat shock promoters, was unable to activate the heat shock response [13,27] (Fig, 4 c and g, circled spots). It was of interest to test whether induction of the acid shock proteins was RpoH-depcndent. Bidimensional electrophoretic patterns of total cellular extracts from strains MA3337 (rpoH +) and MA3334 (rpoH165) exposed to a pH shift were compared (Fig. 4 b and f; Table 1). Heat shock proteins (circled spots 2, 3, 6, 10, 13 and 17) and acid shock protein 72 were not induced by an acid shift in the rpoH165 mutant strain. On the contrary, polypeptide 32 and the other ASP were induced by an acid shift in both wild-type and rpoHI65 mutant strains (Table 1, Fig. 4 b and f, squared and arrow head marked spots).

TaglicM et al. [4] used acid shifts from pH 8.0 to pH 6.0 whereas to induce the acid response, we transfer growing cells from pH 6,9 to pH 4.3. We noticed that a transfer from pH 6.9 ~o pH 5.0 did not induce ASP synthesis. Second, Taglicht ctal. [4] looked for pH shift responses that peaked at 5 to 10 rain as was previously reported for the heat-lnduced response whereas our observations were made 30 to 45 rain after an acid shift. it has been shown that when E. coli cells are transferred from pH 6.4 to pH 4.4, a transient decrease in cytoplasmic pH of about 0.4 unit is observed with recovery within 4 rain [28]. After an acid shift (from pH 6.9 to pH 4.3), kinetics of total protein synthesis might be the direct consequence of cytoplasmic pH variation whereas kinetics of ASP induction should obey a very different mechanism operating after a lag period rather than an emergency mccharfism. Among proteins induced by an acid shift, we have identified 6 heat shock proteins whose induetion by a pH shift-dowr is RpoH.dependent. This result suggests that induction of at least a subset of the so-called heat shock proteins might be an aid for cells to adapt to acidification of the medium. However, the neat shock response per se may be insufficien~ 7 other proteins, which have not been descfib:d previously to be induced by any stress, were actually induced by an acid shift, one of them only being RpoH-dependent. The mechanism by which acidification of extracelluhr pH triggers induction of these proteins is unknown. Preliminary analysis of total cell envelope extracts prepared from acid-shocked cells of E. colt showed that acid shock proteins are nol membrane but soluble components (data not shown).

5. DISCUSSION Previous studies have shown that an alkaline but not an acid shift of extracellular pH activates the heat shock response in E. coli [4]. In this paper, we have shown that heat shock proteins and other previously described stress proteins as well as a subset of previously unknown proteins w e have called acid shock proteins, (ASP) w e r e concomitantly induced by an acid shift. These different observations are in fact associated with different experimental conditions. First,

ACKNOWLEDGEMENTS We are grateful to B, Bachmann for kindly supplying strain K165 We also thank J.C. Cortay for his help in bidimcnsiona[ electrophoresis and F. Crutel and S. Rotmies for expert technical assistance. This work was supported by research grants from the Centre National de la Recherche Sciendfique (UMR106).

26

REFERENCES [1] Neidhardt, F.C. 11957) in Escherichm coli and Salmonella typhimurium Cellular and Molecular Biology {F.C. Neidhardt, ed. in chien, pp. 1313-1317, American Society for Microbiology, Washington, DC. [21 Neidhardt, F,C_, Van Bogeleo. R,A, and Vaaghn, Y, (1984) Annu. Rev. Genet. 18, 295-329÷ [31 Schuldiner, S., Agmon, V., Btandsma, J., Cohen, A.. Friedman, F,,. and Adan, E.P. (1986) J. Bacteriol. 168, 936-939. [4] TagliehL D., Padan, E., Oppenheim, A.B. and Schaldiner, S. (1987) J. BacterioL 169, 885-887. [5] Smith, M.W and Neidhart, F.C. (I983} J, Baetcfiol. I54. 336-343. 16] Winkelman, J,W, and Clark, D.P. (t986} J. Bact¢fiol. 167, 362-367. 17I Smith, M,W. and Neidhart, F.C. 11983) J. Bacteriol. 154, 344-350. 18] Walker G.C. (1984) Mierobiol. Rev. 48, 60-93. [91 Clark, D. and Parker, J. {1984) FEMS Mierobiol. Letl. 25, 81-83. [10] Gutierrez, C., Barondess, J., Manoil, C. and Beckwiih, J. (I987) J. Mol. Biol. 195, 289L297. [11] Wanner, B.L. and MeScharry, R. (1982) J. Mol. Biol. 158, .347-363. [12] Neidhardt, F.C. and Van Bogelen. R.A. (I987) in Escheriehia celi and Sabnonella ryphimurium Cellular and Molecutar Biology (F.C, Neidhardt, ed. in chief), pp. 1334-1345, Amed~:an Society for Microbiology. WashingIon, DC. [131 Neidhardt, F.C. and Van Bogden, R.A. O98I) Biochem, Biophys. Res. Commun. 10fi, 894-900.

[14] Grossman, A.D., Eriekson, J,W~ and Gross. C.A. 11984) Cell 38. 383-3~. [15] Booth, I.R. (19[$5)MJcrobiol. Rev. 49, 359-378. 116] Cooper, S. and Ruetiinger, T. 09"/5) Mol. Gen. Genel. 139. 167-17b. [17] Wanner, B., Kodaira, R. and Ngidhardt, F.C. (1977) J. Bacteriol. IJ0, 212-222. [18J Miller, J. H. 11972) in Experimems in Molecular Genetics, Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. [191 Laemmli. U.K. (1970) Nature (London) 227, 680-685. [20[ O'Farrell, P.H. 11975) J. Biol. Chem. 250, 4007-4021. [21] Cortay, J.C., P,ieul, C.. Duclos, B. and Cozzon¢, AJ, (1986) Eur, J. Biochem. 159. 227-237. 122] Yamamofi. T.. lip, K., Nakamura. Y, and Yura, T. 11978) J. Bacteriol. 134. 1133-1140. [23] Neidhardl, F.C. (1987) in Esekeriehia coh and Salmonella typhsmurium Cellular and Molecular Biology (F.C_ NeidhardL ¢d, in chiefL pp. 919-966, American So,'iety for Microbiology, Washington, DC', [24] Spence, J. and Georgopoulos, C. (19891 J. Biol. Chem. 264, 4398-4403. [25] Krueger, 3.H, and Walker, G,C. (1984) Proc. Nail. Aead. Sci, U.S.A.. 81, 1499-1503. 126] Jones, P.G., Van Boge|en. R.A, and Neidhardt, F.C. (1987) J. Bacleriol. 169, 2092-2095. [27] Yamamori, T. and Yarn. T. 11982) Proc. Nail. Acad. Sci. U.S.A. 79, 860-864. [28l Stonczewski, J.L, Macnab, R.M.. Alger, J.R. and Castle, A,M. 11982) J. Bacteriol, 152. 384-399.