The periplasmic galactose receptor protein of Escherichia coli in relation to galactose chemotaxis

BIOCHIMIE, 1976, 58, 81-85.

The periplasmic galactose receptor protein ot Escherichia coli in relation to galactose chemotaxis (*). H e r m a n M..KAL.CKAR.

Department of Biological Chemistry, Harvard Medical School and Huntington Laboratories Massachusetts General Hospital, Boston, Mass. 0211£ Summary. - - The periplasmic galactose receptor protein of E. colt is the common maoromolecule in the initiation of two functions, ehemotaxis and active transport. The substrates are glucose and galaetose and the affinity for binding to the receptor protein is high (K,,-2 X 10-8 M for glucose and 1 X 10-7 M for galactose). A second binding site shows a 100,.fold lower affinity. The high concentration of the galaetose receptor protein in the periplasmic space tends to give retention through recapture of the ligands. The kinetic properties of the galactose receptor protein are, in general, in harmony with the kinetics of chemotactic responses to spatial or temporal sugar gradients.

INTRODUCTION. In this article to a p p e a r in the m e m o r i a l v o l u m e for Huguette de R o b i c h o n - S z u h u a j s t e r , I greatly a p p r e c i a t e the o p p o r t u n i t y to relate a f e w personal r e m i n i s c e n c e s about o u r association in s c i e n c e d u r i n g the y e a r s 1957 to 1960. This association b r o u g h t me some lasting values, both s c i e n t i f i c and scholarly. If it w e r e not for Huguette, I d o u b t that I Would h a v e gone b e y o n d E n g l i s h t r a n s l a t i o n s of F r e n c h literature. Now, I h a v e taken the time to struggle t h r o u g h c h a p t e r s in F r e n c h f r o m such d e m a n d i n g m o d e r n essays as J a c o b and Monod's epistemologieal discourses in biology. Back in 1958, I r e m e m b e r h o w Huguette persuaded me to glance at a 30,0 y e a r old F r e n c h text by Nicotaus Steno on his p r e c i s e m e t h o d s of disc l o s i n g the s t r u c t u r e s of the b r a i n [1]. Steno discusses the Descartes de Homini m o d e l s and rates t h e m highly, hut r e p u d i a t e s the f o l l o w e r s w h o used t h e m u n c r i t i c a l l y . Huguette studied, of course the o r i g i n a l F r e n c h text by <> and f o u n d it superlative, w h e r e a s the t r a n s l a t i o n she t h o u g h t acceptable but l a c k i n g that e x t r a wit. She p e r s u a d e d me r e a d i l y to s t u d y at ,least some of the h i g h l i g h t s of the old F r e n c h text an,d I did not r e g r e t the time spent. Besides, Steno (Niels Steensen) came f r o m m y h o m e city, Copenhagen. Huguette i n t r o d u c e d me also to some o t h e r relat i v e l y u n k n o w n f r a g m e n t s of F r e n c h s c i e n t i f i c scripts, mosi notab,ly, P a s t e u r ' s first r e p o r t to the (*) Dedicated to the Memory of Dr. Huguette de Robiehon- Szulmaj ster.

Comptes R e n d u s 4e l'Acad6mie des Sciences ¢ le sucre de /nit )> (Lille, 1856) [2]. She r e t r i e v e d that f u n n y ~little gem of w h i c h I m e r e l y cite - - (>. It was w h a t w e n o w call galactose. H o w ever, the m a i n c o n c e r n f r o m P a s t e u r ' s side w a s the n o v e l t y - - this sugar was not just a n o t h e r f o r m of glucose and P a s t e u r felt that it d e s e r v e d a n e w name. F r o m h e r m e n t o r s in F r a n c e , Huguette f o u n d a strong basis in m i c r o b i a l b i o c h e m i c a l genetics w h i c h r e m a i n e d h e r m a i n p a s s i o n in h e r r e s e a r c h . H e r w o r k in o u r l a b o r a t o r y w i t h K i y o s h i Kurahashi, E l i z a b e t h Maxwell and m y s e l f e v o k e d m y interest in m i c r o b i a l b i o c h e m i s t r y , and this inv o l v e m e n t h e l p e d r e o r i e n t m y i n t e r e s t for the next 20 years. I do not i n t e n d to list all Huguette's activities in our l a b o r a t o r y - - let me just m e n t i o n the i m p o r t a n t little opus on the first w e l l docum e n t e d e x c e p t i o n f r o m sequential i n d u c t i o n . This was h e r article in 1958 [3], ¢ I n d u c t i o n of E n z y m e s of the Galactose P a t h w a y in Mutants of Saccharomyces cerevisiae >>. In this b r i e f article, she e x a m i n e d the i n d u c t i o n of the enzymes of the a m p h i b o l i c UDPGal p a t h w a y in a galactosekinaseless m u t a n t of h a p l o i d Saccharomyces cerevisiae (and also in S. fragilis). I n d u c t i o n by free galactose of step 2 and 3 in the p a t h w a y (i.e. Gal-l-P u r i d y t trans~erase a n d UD~PGtucose 4-epimerase) p r o c e e d e d d r a m a t i c a l l y in spite of the m i s s i n g step 1 and its p r o d u c t (Gal-l-P). This is a differ e n t situation f r o m that of s e q u e n t i a l i n d u c t i o n . Huguette also d e m o n s t r a t e d t'hat the e p i m e r a s e in yeast (S. cereoisiae, S. fragilis) is not constitutive in spite of the p r e s e n c e of UDPGlucose in the cells [4]. 6

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I n galactokinase-less m u t a n t s of Escherichia colt, the n o n - s e q u e n t i a l type of i n d u c t i o n can also he demonstrated, although the difference b e t w e e n the n o n - i n d u c e d ~and the i n d u c e d levels of the next two enzymes is less d r a m a t i c because of the relatively high unindu.ced levels i n E. colt [5]. Nevertheless, these galactokinase-less E. colt K-12 strains r e m a i n e d ahnost a sort of s t a n d a r d refer e n c e also in our r e c e n t studies on gala ctose transp o r t an,d the correspondin~g galactose chemotactic protein. These studies were c a r r i e d out by W i n f r i e d Boos who after his six years i n our l a b o r a t o r y j o i n t e d Maxime Schwartz at the Institut P a s t e u r a n d n o w is h e a d i n g a b i o c h e m i s t r y d e p a r t m e n t at the U n i v e r s i t y of Konstanz. A study of the w o r k b y Julius Adler [61 a n d his coworkers on the gen,etics o*f t r a n s p o r t a n d chemotaxis caught our interest. We were especially interested i n the m u t a n t s w h i c h showed defects of galactose transport, yet h a d p r e s e r v e d galactose chemotaxis [7]. This c o m b i n a t i o n seereed r e n l i n i s c e n t of some of our transport-defective m u t a n t s w h i c h still synthesized p l e n t y of the p e r i p l a s m i c galactose b i n d i n g p r o t e i n [:8, 9]. I therefore venture,d to correlate the existence of galacrose chemotaxis w i t h the p r e s e n c e of a f u n c t i o n a l galactose b i n d i n g p r o t e i n [81 and Hazelbauer a n d Adler p r o v e d the p o i n t [9]. I w a n t to discuss some k i n e t i c p r o p e r t i e s of the galactose bindin,g p r o t e i n as described b y W i n fried Boos, since some o~ these p r o p e r t i e s seem so p e r t i n e n t to the p r o b l e m of chemotaxis. Before d e l v i n g into these problems, let me just sketch briefly w h a t is c o n s i d e r e d the essence o~ chemotaxis a c c o r d i n g to Adler and H o w a r d Berg. Berg, wtio has been able to trace single b a c t e r i a i n gradients of various attractants (and the t r a c i n g is 3-dimensional, m u c h like an a s t r o n o m e r ' s approach) describes two m a i n phases of t h e i r motion, t w i d d l i n g (or .tumbling) and s w i m m i n g . If the b a c t e r i u m h a p p e n s to s w i m up the g r a d i e n t of an attractant, the tveiddling is s u p p r e s s e d significantly [10]. If the vector of the d i r e c t i o n oq swimm i n g is zero or negative (down the gradient) the i n c i d e n c e of t w i d d l i n g is not suppressed. I n temporal gradients, the outcome, a c c o r d i n g to Berg, is essentially the same [111. A c c o r d i n g to Macnah a n d Kosh'land, s u d d e n and large negative p e r t u r b a t i o n s i n c o n c e n t r a t i o n s of attractants can elicit an, i n c r e a s e d inciden,ce of t w i d d l i n g over a n d above that seen at a c o n s t a n t c o n c e n t r a t i o n of the attractants [12]. However, if c o n c e n t r a t i o n s changes are held w i t h i n the o r d e r of m a g n i t u d e of that of the dis-

BIOCHIMIE, 1976, 58, n ° 1-2.

sociation constant of the b i n d i n g protein, the prev a i l i n g observable response is that of s u p p r e s s i o n of the t w i d d l i n g , thus r e s u l t i n g in <(c l i m b i n g >) a g r a d i e n t of an a t t r a c t a n t [10, I1]. How does the kinetics of sugar chemotaxis correlate w i t h that of the bin,ding p r o t e i n ? Since the emphasis in the following discussion will c e n t e r a r o u n d c h e n m t a c t i c responses to s e n s o r y stimuli, let me r e n a m e the galactose hin~ding protein, (( the galactose receptor p r o t e i n )~ i n analogy w i t h other s e n s o r y receptors.

I~ESULTS AND DISCUSSION. A c o m p a r i s o n of substrate specificity i n terms of i n h i b i t i o n of b i n d i n g of galactose to its perip l a s m i c receptor p r o t e i n or the i n t e r f e r e n c e of galactose chemotaxis by various sugars, shows a r e m a r k a b l e correlation (see table I, [9J). TABLE I.

Specificity of Gal chemoreceptors and Gal binding protein [after Hazelbauer a n d Adler, 1971]. Concentration (,uM) required Ior 50 p. cent inhibition Interceptors

(A~

(g) Bin~

Taxis l~Mof Ratio toward B/A 5u.M galac~alaetose tose D-glucose . . . . . . . . . . . . . . . . . D-galaetose . . . . . . . . . . . . . . . . 1- D-glycerol-~-D-galactoside D-fUcose . . . . . . . . . . . . . . . . . . . ,~-Megal . . . . . . . . . . . . . . . . . . . L -arabinose . . . . . . . . . . . . . . . . D-xylose . . . . . . . . . . . . . . . . . .

0.005 1.0 0.036 7.0 0.15 112500 6.2 30 3500 95 h17,000 120 118,000

200 190 170 180 120 180 150

A Scatchard plot of data o b t a i n e d front equil i b r i m n dialysis [with the gala ctose r e c e p t o r protein in c o n c e n t r a t i o n s not exceeding 0.3 to 0.4 rng p e r ml [13] showed a n u m b e r of i n t e r e s t i n g features (see fig. 1). 1) There are two b i n d i n g sites for galactose per m o n o m e r r e c e p t o r protein, a m e d i u m affinity site of a K D of 10 5 M a n d a high affinity site of a K D of l i p 2 × 10 -7 M [13]. The Scatchard plot i n r e v e r t a n t s (or p s e u d o r e v e r t a n t s ) from low-affinity m u t a n t s show the same features [141. 2) There is a <(t a i l i n g >> of the plot t o w a r d s the t e r m i n a l region ~vhere the m o l a r c o n c e n t r a tions of receptor sites greatly exceeds that of

The periplasmic galactose receptor of E. colt. galactose. The i n t e r p r e t a t i o n of the tail e n d is not, as yet, clear. The galactose r e c e p t o r p r o t e i n has a high affinity for free glucose or galactose ; the K D of the latter is, as m e n t i o n e d , of the o r d e r of 1 X 10 7 M [13] an,d K D for glucose is p r o b a b l y of the order of 0.2 to 0.3 X 10 -7 M [9]. 20 18 16 14

\

~10 ~ 0s &

\ ~,4h

a

O2

~,~_,~_~a_ % ~ _ _ ^ . ~ , ~ _

00 i 1

I J I I I [ I I I I I 2 3 /, 5 6 7 8 9 10 11 12 bound galactoseltotal prolevi x free galactose concentration (X10-5)

i]3

FIG. 1. - - Scatchard plot of galactose-binding activity of the galactose receptor protein measured by equilibrium dialysis for at least 12 hours (protein concentration app. 1 X 10-5 M) [after Boos et al., ~13].

If the high affinity receptor p r o t e i n is p r e s e n t in c o n c e n t r a t i o n s exceeding 1 mg per ml or 25 to 30 ~xM, the t e n d e n c y of r e t e n t i o n of galactose is very high [15, 161 a n d the dissociation of the specific ligand becomes a rare event [16]. Since glucose shows very high affinity for the galactose receptor protein, b o u n d glucose is strongly retained [16] as if it were eovalentay b o u n d to the p r o t e i n [17]. Yet, it is merely a m a n i f e s t a t i o n of r e c a p t u r e [16]. The same features have been f o u n d r e c e n t l y for the maltose r e c e p t o r p r o t e i n a n d maltose or maltotriose [18]. The E. colt cell m a y c o n t a i n as m a n y as 30,000 to 50,000 galaetose receptor p r o t e i n s on the outer surface of the i n n e r m e m b r a n e [15, 19], a n d h e n c e the chance for r e c a p t u r e of galactose molectdes released from cellular receptors w o u l d be very high. This, in turn, m e a n s that the n u m b e r of ligands dissociating per time u n i t is relatively very low. W h a t does this m e a n in terms of function ? A c c o r d i n g to Berg's analysis of bacterial behavior in m o d e r a t e t e m p o r a l or spatial gradients of attraetants, a chemotactie response i.e., a bias i n favor of straight s w i m m i n g , is only elicited up the g r a d i e n t [11]. If r e c a p t u r e of galaetose is a m u c h more likely event t h a n dissociation, w o u l d the ¢ sluggishness

BIOCH1MIE, 1976, 58, n ° 1-2.

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of i n f o r m a t i o n >> then become d i s t u r b i n g l y high, especially for a response to negative g r a d i e n t ? In a joint project from Sehwartz's u n i t (Silhavy

et al., [18]), the r e t e n t i o n of maltose or maltotriose by the maltose receptor p r o t e i n has been subjected to a theoretical q u a n t i t a t i v e t r e a t m e n t of general interest. In this c o m p u t a t i o n , it was clearly d e m o n s t r a t e d w h y extreme r e t e n t i o n of l i g a n d s can be expected if the affinity to the receptor is high a n d the m o l a r i t y of u n f i l l e d receptors exceeds that of the l i g a n d [18]. A c c o r d i n g to this c o m p u t a t i o n a n d an assessment of galactose receptor p r o t e i n s in the p e r i p l a s m i e space, one could p r e d i c t that b a c t e r i a w o u l d r e s p o n d m u c h more r a p i d l y to positive gradients of galactose t h a n to negative. We shall, however, confine ourselves largely to a discussion of positive gradients a n d the characteristics of the p e r i p l a s m i c receptor p r o t e i n s a n d h o w they correlate w i t h responses to spatial or t e m p o r a l gradients as described by the Adler a n d Berg laboratories, w i t h special emphasis on sugar chemotaxis. The recent study from I n s t i t u t Pasteur on the maltose (and maltotriose) r e c e p t o r p r o t e i n has become of a d d i t i o n a l interest since this p r o t e i n seems to he i n v o l v e d in maltose chemotaxis [20]. I n galactose as well as maltose chemotaxis, the chemotactic response reaches the peak of its sensitivity at very low c o n c e n t r a t i o n s of ligand. I n the case of galactose, Mesibov, Ordal a n d Adler found a chemotactic K~ at 6 X 10 -7 M [21]. In such a c o n c e n t r a t i o n range and w i t h i n the volume of b a c t e r i u m , one w o u l d have m a n y more empty receptor sites than ligands. The fluctuations of the n u m b e r of ligands w i t h i n such a small volume might well range b e t w e e n 101 a n d zero. Chemota.ctic responses to such r a p i d r a n d o m f l u c t u a t i o n w o u l d make the b e h a v i o r of the cell very erratic even in a steep positive gradient, if a d a m p i n g feature of r e t e n t i o n d i d not m a k e itself felt. The r e t e n t i o n p h e n o m e n o n is therefore b e i n g cited quite correctly as an i n t e g r a t i n g feature i n the chemotactic response to gradients of attractant [18]. The r e t e n t i o n o.f dissociation from the p e r i p l a s m i c galactose receptor p r o t e i n m a y well exceed a factor of 104 [18J. This might c o r r e s p o n d to a time delay of 0.1 to I second, d e p e n d i n g o n the n u m b e r of receptors p e r A2 a n d on the effect of the outer m e m b r a n e (H. Berg, p e r s o n a l c o m m u nication). The speed of straight r u n s is a r o u n d 20 bacterial b o d y lengths p e r second [10]. A delay of 0.1 to 1 second c o r r e s p o n d i n g to an average of about 10 body lengths (or 10 ~aneters) seems therefore a useful feature for a response w h i c h b r i n g s

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H. M. K a l c k a r .

the b a c t e r i u m to m o v e up the g r a d i e n t of an a t t r a c t a n t by a ¢ b i a s e d r a n d o m w a l k >> [11]. T h e i n f o r m a t i o n f r o m the p e r i p l a s m i c galactose r e c e p t o r c o m p l e x is s u b s e q u e n t l y b e i n g transm i t t e d to a m e m b r a n e r e c e p t o r w h i c h O r d a l a n d A d l e r h a v e called a <~signaller >> [22]. Genetic e v i d e n c e i n d i c a t e s that the <> r e s p o n d s both to galactose r e c e p t o r c o m p l e x e s as well as to ribose r e c e p t o r c o m p l e x e s [22]. The next step in the s e n s o r y s e q u e n c e is presum a b l y the so-called t w i d d l e (or rustable) generator. , Its b i o c h e m i c a l c h a r a c t e r i s t i c s are b e i n g e x p l o r e d a n d little can be said at present. H o w e v e r , Spud i c h an.d K o s h l a n d [23] h a v e r e c e n t l y p u b l i s h e d an i n t e r e s t i n g a n d i m p o r t a n t study of the r e l a t i o n ship b e t w e e n r e c e p t o r o c c u p a n c y and flagella r e s p o n s e (**). T h e c h a p t e r s on the m e c h a n i s m of flagella f u n c t i o n of straight s w i m m i n g or of t u m b l i n g (by the ~( t w i d d l e g e n e r a t o r >>) constitute an extensive t o p i c by itself w h i c h is w e l l d e s c r i b e d by A d l e r [24]. A b r i e f discussion should be a d d e d on the role of the galactose r e c e p t o r p r o t e i n in active "transp o r t of galactose. A h i g h l y active E. colt transp o r t system of galaclose has been d e s c r i b e d first by H o r e c k e r , T h o m a s and Monod [25] a n d later the genetics o'f the r e g u l a t i o n of the system was s t u d i e d [26]. T h e d i s c o v e r y of the p e r i p l a s m i c galactose b i n d i n g p r o t e i n [27] r a i s e d the p r o b l e m ~f its role in the active t r a n s p o r t o,f galactose. This has been dis, cussed in d e p t h in a r e c e n t r e v i e w by Boos [28]. It m a y be sufficient to state h e r e that the galactose r e c e p t o r p r o t e i n serves not only as the first step, but also c o n v e y s h i g h affinity of galactosc t r a n s p o r t [28]. As to the t r a n s p o r t f u n c t i o n of the galactose r e c e p t o r protein, Boos and his c o w o r k e r s have c l a r i f i e d a n u m b e r of i m p o r t a n t points [15, 19, 28]. The galactose r e c e p t o r p r o t e i n functio,ns presumably : (i) as a high affinity r e c e p t o r for glucose a n d galactose ; (it) as a r e c e p t o r c o m p l e x w h i c h elicits a change in s u r f a c e charge ( a p p e a r a n c e of a net n e g a t i v e charge) ; (**) Their description of responses of the riboseallose receptor in Salmonella is, essentially, in accordance ",vith the present disettssiort. Any delay in the theoretical time schedule of a recovery response due to recapture could only be expected in the concentration range 10-s to 10-7 M ribose and might be difficult to pick up unless the retention delay approached 1 sec, and the recovery response were determined by negative concentration jumps. BIOCHIMIE, 1976, 58, n ° 1-2.

(iii) as a p a r t of t r a n s p o r t system w h i c h b i n d s to the i n n e r m e m b r a n e c o n t a i n i n g the galactose p e r m e a s e . The galactose r e c e p t o r p r o t e i n c o n f e r s h i g h affinity to e n t r a n c e of galactose a n d h e n c e is a n e c e s s a r y l i n k in the e n e r g y - d r i v e n ability to c o n c e n t r a t e galactose against steep g r a d i e n t s ( e x c e e d i n g 5 X 10a). Although the galactose r e c e p t o r p r o t e i n therefore seems an integral p a r t of a c t i v e t r a n s p o r t of galactose, there is m o u n t i n g e v i d e n c e that it does not f u n c t i o n d i r e c t l y in t r a n s l o c a t i o n (see [28]). M e m b r a n e vesicles f r o m E. colt are able to c a r r y out a low-affinity yet g e n u i n e i r a n s l o c a t i o n of galactose [29]. Genetic e v i d e n c e p r e s e n t e d r e c e n tly by Ord'al and A d l e r [22] and by R o b b i n s a n d R o t m a n [30j i n d i c a t e s that t w o genes are r e q u i r e d for t r a n s l o c a t i o n and a t h i r d one in a d d i t i o n f o r the m a r k e d a c c u m u l a t i o n of gal.actose, c h a r a c t e r istic for E. colt strains possessing the p e r i p l a s m i c galactose r e c e p t o r p r o t e i n P a r n e s and Boos [19] h a d a l r e a d y s h o w n that the galactose r e c e p t o r p r o t e i n is not i n v o l v e d in exit but only in entry. Yet, exist of p r e a c c u m u lated gala.ctose can be s t r o n g l y s t i n m l a t e d by a d d i t i o n to the m e d i u m of s p e c i f i c substrates for the r e c e p t o r . This so-called <> slim u l a t i o n of exit needs t h e r e f o r e the p e r i p t a s m i c c o m p o n e n t as well as the m e m b r a n e c o m p o n e n t [19]. This is yet a n o t h e r d e m o n s t r a t i o n of the integrated, complex, and h i g h l y active galactose t r a n s p o r t system of E . c o l i . Acknawledgments. I am thankful for fruitful discussions with Drs. J. Adler, H. Berg and W. Boos. This work was supported by grants from U.S. Public Health Service, NIH (AM 05507), National Science Foundation and American Cancer Society. R~su~r~. La prot6ine p6riplasmique affine du galactose cbez E. colt est responsable de la chimiotaxie et du transport actif. Ses substrats sont le glucose et le galactose, dont les activit6s respectives sont de 2 X 10-s M et I × 10-7 M. Un denxi6me site de liaison poss6de une affinit6 100 fois infdrieure. La concentration 61ev6e de la prot6ine r6eeptrice de l'espace p6riplasmique tend h produire une r6tention par recapture du ligand. Les propri6t6s ein6tiques de la prot~iue rdcep,trice sont en accord avee les r6ponses ehimiotaetiques aux gradients spatiaux ou temporels des sucres, REFERENCES. 1. Discours de Monsieur Stenon sur l'Anatomie du Cerveau (A Messieurs de l'Assembl6e qui fait chez Monsieur Thevenot) h Paris (chez Robert de Ninville) MDCLXIX (avec PriviI6ge du RoD. Reprinted Copenhagen 1950, Nyt Nordisk Forlag.

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