Cation Transport in Escherichia coli VIII. Potassium Transport Mutants D A V I D B. R H O A D S , F R E D B. W A T E R S , and W O L F G A N G E P S T E I N From the Departments of Biochemistry and of Biophysicsand Theoretical Biology, University of Chicago, Chicago, Illinois 60637

ABSTRACT Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 p.M, and Vmax at 37°C of 150/zmol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have K,,'s of 1.5 and 0.5 mM and Vm~x'sof 550 and 40 at 37 and 30°C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 raM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells ofE. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.

Cells o f the g r a m - n e g a t i v e b a c t e r i u m Escherichia coli s h a r e with most o t h e r cells the p r o p e r t y o f m a i n t a i n i n g m u c h h i g h e r i n t r a c e i l u l a r c o n c e n t r a t i o n s o f K t h a n those in the e x t r a c e l l u l a r m e d i u m . I n t r a c e l l u l a r K in E. coli is d e t e r m i n e d l a r g e l y by the o s m o l a r i t y of the e x t e r n a l m e d i u m (Epstein a n d Schuhz, 1965; E p s t e i n a n d Schultz, 1968) a n d is virtually i n d e p e n d e n t o f the c o n c e n t r a t i o n o f K in the e x t e r n a l m e d i u m (Schultz a n d S o l o m o n , 1961). I n t r a c e l l u l a r K r a n g e s f r o m 150 mM in cells g r o w n in 80 mosM m e d i a to n e a r l y 600 mM in cells g r o w n in 1,200 mosM m e d i a (Epstein a n d Schultz, 1968). Osmotic r e g u l a t i o n o f i n t r a c e l l u l a r K also occurs in Salmonella ( C h r i s t i a n , 1955) a n d m a y be c h a r a c t e r i s t i c o f m a n y genera of bacteria. Cells o f E . coli K-12 are c a p a b l e o f high rates o f K u p t a k e . Such u p t a k e has a Km for e x t e r n a l K o f a p p r o x i m a t e l y 5 mM when m e a s u r e d in cells d e p l e t e d o f K by g r o w t h into late s t a t i o n a r y p h a s e (Schultz et al., 1963). R a p i d r a t e s o f K u p t a k e ~:an be p r o d u c e d in e x p o n e n t i a l p h a s e cells by a s u d d e n i n c r e a s e in o s m o l a r i t y (Epstein a n d Schultz, 1965). U n d e r these c o n d i t i o n s the K,, f o r u p t a k e is a p p r o x i m a t e l y 1 m M . I n b o t h cases m a x i m u m rates are h i g h , r a n g i n g f r o m 130 to 2 4 0 / z m o l / g min at 30 a n d 37°C, respectively. U n d e r both c o n d i t i o n s T H E , J O U R N A L OF G E N E R A L P H Y S | O L O G Y " V O L U M E

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the major ionic m o v e m e n t maintaining electroneutrality d u r i n g K uptake is p r o t o n extrusion. In stationary phase cells which have elevated pools of Na, part of the K taken up is e x c h a n g e d for Na (Schultz et al., 1963). In o r d e r to gain a more detailed knowledge o f the process o f K transport, mutants affecting this process were isolated. All the mutants studied to date were isolated by m e t h o d s that enrich for mutants requiring h i g h e r K concentrations for growth than n e e d e d by the wild type. T h e mutants were isolated in two steps. Beginning with a wild-type strain and using mild ultraviolet mutagenesis to minimize the incidence o f multiple mutations, only a single class o f mutants was obtained (Epstein and Davies, 1970). These strains have mutations in any one o f four closely linked kdp genes. T h e kdp mutants require 0.07 mM K to achieve a half-maximal rate o f growth, differing f r o m the wild type which has a constant rate of growth down to concentrations o f 5 #M or less (Weiden et al., 1967). Mutants r e q u i r i n g more K for growth than the kdp mutants were not obtained by mutagenizing wild-type strains, but were readily obtained by mutagenizing a kdp m u t a n t strain. In this way five types o f double mutants were identified (Epstein and Kim, 1971), each carrying the original kdp mutation and a f u r t h e r mutation in five o t h e r genes labeled trkA t h r o u g h trkE. Each class was distinguished by growth tests on plates, aDd was shown to be due to a mutation in a distinct gene. T h e five trk genes are widely scattered on the E. coli c h r o m o s o m e , n o n e lying extremely close to any o t h e r or to the kdp genes, although two (trkA and trkB) are close e n o u g h to be c o t r a n s d u c e d by Plkc. We here describe the kinetics o f K t r a n s p o r t in the mutants. All of the mutants differ from the wild type in K transport. T h e data below indicate that E. coli has three saturable K uptake systems, called the Kdp, T r k A , and T r k D systems by analogy with the genetic symbols for the genes affecting these processes. A fourth system which is not saturable, T r k F , appears to be present in all mutants. Two genes, trkB and trkC, are associated with defects in K retention. A preliminary r e p o r t o f some o f this work has been presented (Epstein, 1970). METHODS

Bacteria The principal bacterial strains, all Escherichia coli K-12, are listed in Table I. The origin of parental strain FRAG-I and the isolation of most of the mutants derived from FRAG-1 have been described (Epstein and Davies, 1970; Epstein and Kim, 1971). For convenience in referring to the different types of K transport mutants the abbreviations listed in the first column of Table I are used. A capital letter followed by a superscript minus sign refers to a defect in a single class of genes; K refers to a mutation in the kdp genes, and letters A- through E- refer to mutations in the trkA through trkE genes, respectively. Thus a strain listed as K-A- has mutations in the kdp and trkA genes, and other genes affecting K transport are wild type. The symbols Kdp, TrkA, and TrkD refer to the transport systems associated with the corresponding genes, while TrkF refers to a nonsaturable transport system apparently present in all of the mutants and seen clearly in mutants lacking the three saturable systems. No mutations affecting the TrkF system have been identified. The derivative strains listed in Table I were obtained by Plkc-mediated transduction, taking advantage of the different K requirements of the mutants. K-D- strain TKI001

RHOADS, WATERS, AND EPSTEIN

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K Transport Mutants of E. coli TABLE

I

BACTERIAL STRAINS Genotype Mutant class*

Typical strain(s):~ K transport related

Wild type K- (Trk +) K-A (TrkA) K-B- (TrkB) K-C- (TrkC)

FRAG-1 FRAG-5 TK133 TK1 i0 TK118 TK121

kdpABC5 kdpABC5 trkA133 kdpABC5 trkBllO kdpABC5 trkC118 kdpABC5 trkC121

K-DK-E- (TrkE) K-A-D- (TrkA/D)

TK1001 TKI42 TK401 TK1002

kdpABC5 trkD1 kdpABC5 trkE142 kdpABC5 trkA401 trkDl kdpABC5 trkA133 trkDl

AA-D-

TK 1005 TK1004 TK1030

trkA133 trkA401 trk D1 trkA401 trkD1

Other§

malA r~adA

* Symbols in parentheses are those used in Epstein and Kim, 1971. 1: The prefix 2K was used in place of TK in referring to these strains in Bhattacharyya et al., 1971. § All strains also have gal rha lacZ and thi mutations (Epstein and Kim, 1971). was obtained as a transductant of TK401 capable of growth in m e d i u m containing 0.1 m M K but not in m e d i u m containing no added K (K0 m e d i u m , see below). T h e A - and A - D strains were obtained by transduction to growth on K0 plates o f the c o r r e s p o n d i n g parent carrying a kdp mutation. Derivatives carrying the FI00 and FI41 episomes (previously designated Flgal and F-41, respectively) were p r e p a r e d essentially as described earlier (Epstein and Davies, 1970; Epstein and Kim, 1971). T r a n s f e r of F100 was selected for by the nadA marker, which was kindly provided by Gerald Tritz in strain UTH4679 and introduced into our strains by cotransduction with the gal marker. T h e nadA locus is cotransduced with kdp at a frequency of approximately 20%.

Media and Growth o f Bacteria In most experiments the phosphate-buffered media described earlier (Epstein and Davies, 1970; Epstein and Kim, 1971) were used. These are r e f e r r e d to by K concentration in millimolar; K0 contains no ad d ed K, while KI15 contains 115 mM K. T h e small amount o f K, usually between 5 and 20 lzM, contaminating K0 m e d i u m had to be removed for studies with the Kdp system. Accordingly what is referred to as K-free medium was p r e p a r e d by incubating K-starved wild-type E. coli in K0 m e d i u m containing glucose for approximately 30 min at 30°C, a time sufficient to allow the cells to remove all but traces o f K (see Fig. 4 below and Epstein and Davies, 1970). T h e bacteria were then removed by filtration through 0.45-/xm pore size m e m b r a n e filters (Millipore, type HA). A few experiments were done with maleate-buffered media containing: 8 mM (NH4)2SO4, 0.4 mM MgSO4, 6 #M FeSO4, 1 mM Na2HPO4, 5 mM Na citrate, 3 g,M thiamine HC1, and either 60 mM of K, Na, or Tris maleate, or 90 mM Mg maleate. T h e desired K concentration was attained by mixing sufficient K maleate m e d i u m with one of the other maleate media. Except as noted below, cells were grown with agitation at the same t e m p e r a t u r e as was to be used for transport measurements, and glucose at 2 g/liter was present d u r i n g both growth and subsequent steps. Cells were harvested in the exponential phase o f growth

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except for cells subjected to K limitation to derepress the Kdp system. T h e latter treatment was accomplished either by growing cells in low K medium, usually K0.05, for approximately 2 h after the time the cells had exhausted the medium of K, or by transferring cells from the growth m e d i u m to K0 containing glucose and agitating for a further 2 h.

Transport Studies A good steady state for transport Work is achieved by the simple expedient of suspending cells in a solution similar to growth medium and containing glucose but lacking a nitrogen source. Such solutions, referred to by the suffix NO after the K designation of the medium (KON0 contains neither K nor NH4), were prepared by substituting osmotically equivalent a m o u n t s of NaC1 for the (NH4)2SO4 in growth medium. Chloramphenicol, 50 ms/liter, was added to inhibit protein synthesis. Wild-type cells maintain constant cell K for several hours when incubated with aeration in such solutions. To stimulate net K uptake the cells must either be depleted of K, or subjected to osmotic upshock (Epstein and Schuhz, 1965). In most experiments K was depleted by a 30-rain treatment with 10 mM 2,4-dinitrophenol (DNP) in KON0 lacking glucose. This concentration of DNP removes over 90% of cell K, most of the loss occurring in the first minutes. T h e cells were then washed and incubated in KON0 for approximately 30 rain before transport was measured. Extensive spontaneous loss of K occurs in certain of the mutants when suspended in KON0 (Fig. 1). This method of K depletion was used in some experiments. After incubation until the cells achieved a new, lower steady state, the cells were washed and suspended in fresh KON0 for transport measurements. Uptake was initiated by adding K to the desired concentration. K concentrations up to 10 mM were achieved by adding KC1; in most cases compensatory amounts of NaCI were added so that the same osmolarity was maintained at all K concentrations. To achieve higher K concentrations, the cells were concentrated approximately 10-fold and added to tubes containing suitable mixtures of K115N0 and KON0. For studying transport by the osmotic upshock method, cells were grown in dilute medium consisting of K10 or K115 diluted with an equal volume of water. T h e cells were washed and transferred to similarly dilute KON0, and net K uptake was initiated by adding 4 vol of cell suspension to 1 vol 2.5 M glucose in dilute NO solution containing five times the desired final K concentration. Cell samples were collected on gridded 0.45-~m pore size m e m b r a n e filters (Millipore), washed briefly with 0.4 M glucose (0.8 M glucose for the upshock experiments) containing 1 mM Tris CI (pH 7), dried, and analyzed for K with an internal standard flame photometer (Instrumentation Laboratory, Inc., Lexington, Mass., model 143). K uptake u n d e r the conditions described above is linear until the cells have taken up approximately one-half of the K they will ultimately take up, after which time net uptake progressively slows down. All rates measured are initial rates. The inclusion of Tris Ci in the wash solution and more extensive washing made it possible to obtain reliable measurements of cell Na by the filter method. It was necessary to wash most batches of filters with 10 mM NH4C1 and then water to avoid very high and rather variable filter blanks for Na. Due to the large a m o u n t of Na in the media and the relatively small amounts in the cells, errors due to trace contamination of cell samples with medium are much larger than is the case for K samples. Transport rates are expressed as micromoles taken up per minute and gram dry weight. T h e latter was estimated from measurements of the turbidity of the cell suspensions in a Bausch and Lomb Spectronic 20 colorimeter (Bausch & Lomb, Inc., Rochester, N.Y.) and a calibration curve for that instrument. This method is less precise than the

RHOADS, WATERS, AND EPSTEIN K Transport Mutants of E. coli

329

direct measurements of pellet volume used earlier (Epstein and Schuhz, 1965) but is much more convenient. Variance in this way of measuring transport affects our estimates of Vmax but does not affect the Km determinations, each of which is based on measurements .of different samples of the same cell suspension. Previously published data relating cell water and cell surface area to dry weight (Schultz and Solomon, 1961) were used to convert filter data for cell K and Na to concentrations and to express the earlier flux rates mentioned in the introduction from units of picomoles per square centimeter second to the units used in this paper. 1 pmol/cm 2 s corresponds to 12.9 ~mol/g min. Growth rates were measured as described earlier (Epstein and Davies, 1970). For such studies cells were grown to midexponential phase in medium of the same type as to be used but containing the highest K concentration attainable in that medium. After washing, the cells were inoculated to a density of approximately 108 cells/ml in a series of 18 x 150-ram tubes containing media of different K concentrations and shaken at 37°C. Turbidity was measured at intervals in a Bausch and Lomb.Spectronic 20 colorimeter, and the growth rate was measured over the interval where the logarithm of the turbidity increased linearly with time. All chemicals used were reagent grade. 42K was obtained from New England Nuclear Corp., Boston, Mass. RESULTS

An initial s c r e e n i n g o f the m u t a n t s was p e r f o r m e d by e x a m i n i n g the rate at which K is lost f r o m cells s u s p e n d e d in KON0 c o n t a i n i n g glucose a n d c h l o r a m p h e n i c o l . T h e results a r e shown in Fig. 1. T h e K - m u t a n t loses very little K in 2 h a n d in this r e g a r d is similar to t h e w i l d - t y p e a n d K - D - m u t a n t s which lose litde, if any, K u n d e r these c o n d i t i o n s (data n o t shown). T h e o p p o s i t e e x t r e m e is seen in the K - B - a n d K - C - m u t a n t s , which lose K v e r y r a p i d l y . B e t w e e n these e x t r e m e s a r e the t h r e e o t h e r m u t a n t classes, all o f which lose K r a t h e r slowly. T h e K - A - D - m u t a n t s e v e n t u a l l y lose a l m o s t all o f t h e i r K, r e t a i n i n g less t h a n 10% after 2 h at 37°C. T h e d i f f e r e n c e b e t w e e n the two r a p i d loss m u t a n t s a n d the o t h e r s is even g r e a t e r t h a n s u g g e s t e d by the d a t a o f Fig. 1 because the r a p i d loss m u t a n t s were e x a m i n e d at 25°C to p e r m i t a m o r e a c c u r a t e d e t e r m i n a t i o n o f the rate, while the o t h e r s (except f o r the K - strain) were e x a m i n e d at 37°C. A way o f a n a l y z i n g the results is to a s s u m e t h a t m u t a n t s d e f e c t i v e in u p t a k e will lose K only as fast as it n o r m a l l y leaves the cells. T h e n o r m a l exit r a t e can be e s t i m a t e d f r o m the r a t e at which K e x c h a n g e s in the s t e a d y state, which occurs with a h a l f time o f a p p r o x i m a t e l y 20 min at 30°C in wild-type strains (Epstein a n d Schultz, 1966). By this c r i t e r i o n the K - B - a n d K - C - m u t a n t s have a d e f e c t in K r e t e n t i o n since they lose K with a h a l f t i m e o f 7.5 a n d 3.5 m i n , respectively. All o f the o t h e r m u t a n t s lose K with a h a l f time e x c e e d i n g 40 rain a n d w e r e t h e r e f o r e s u s p e c t e d o f h a v i n g defects in K u p t a k e . M e a s u r e m e n t s o f K u p t a k e c o n f i r m e d the classification i n f e r r e d f r o m the d a t a in Fig. 1. We have i d e n t i f i e d f o u r s e p a r a t e K t r a n s p o r t systems in E. coli whose p r o p e r t i e s a r e s u m m a r i z e d in T a b l e I I . T h e kinetic p r o p e r t i e s a r e f r o m d a t a p r e s e n t e d below; the genetic p r o p e r t i e s are f r o m p u b l i s h e d d a t a (Epstein a n d Davies, 1970; E p s t e i n a n d Kim, 1971). T h e lowest rates o f t r a n s p o r t a r e seen in the t r i p l e K - A - D - m u t a n t s which lack all t h r e e s a t u r a b l e u p t a k e systems. T r a n s p o r t rates in such strains a r e

330

THE JOURNAL OF GENERAL P H Y S I O L O G Y ' V O L U M E 67- 1976 L

IOC

K 18 c

?,

8C 12

60 W v

"6 ~ 4O

13. D v

6

i,

ILl

0

o~

g

,'o"~o

do

,6o

TIME (rnin) FIGURE

1

,~o

o~

4'o

,~o

go

K CONCENTRATION

(raM)

FIGURE 2

FIGURE 1. Spontaneous loss o f K after suspension in K0 buffer. Cells in the exponential phase of growth were collected and washed by filtration, and susp e n d e d at a density o f approximately 250 ~.g/ml in KON0 containing 2 mg glucose and 50/~g chloramphenicol p e r milliliter. At the indicated times samples were taken for m e a s u r e m e n t of intracellular K. @, K- strain FRAG-5, t e m p e r a t u r e 25°C; ©, K - E - strain TKI42, 37°C; A K - A - strain TKI33, 37°(]; V, K A - D - strain TKI002, 37°C; [3, K - B - strain TKI10, 25°C; O, K C- strain T K I I 8 , 25°C. Note change in scale of abscissa after 10 rain. FIGURE 2. Dependence of K uptake rate on K concentration in K - A - D - mutants at 37°C. Data fl)r three separate experiments are shown. Exponential phase cells of strain TK1002 (O, El) or strain TK401 (A) were depleted o f K by incubation in KON0 buffer containing glucose and chloramphenicol for 130 rain ([], A) or 180 rain (Q). T h e n the cells were concentrated by filtration and a d d e d to a series of tubes containing mixtures of KON0 and K115N0 to achieve the indicated final K concentrations. T h e initial rate of uptake at each concentration is plotted. l i n e a r l y d e p e n d e n t o n e x t e r n a l K c o n c e n t r a t i o n u p to 105 m M , t h e h i g h e s t c o n c e n t r a t i o n t e s t e d (Fig. 2). D a t a f o r t h r e e e x p e r i m e n t s a r e s h o w n . I n e a c h case l i n e a r i t y is s e e n , a l t h o u g h t h e a b s o l u t e v a l u e s d i f f e r e d s o m e w h a t in o n e o f t h e e x p e r i m e n t s . I f this u p t a k e p r o c e s s is s a t u r a b l e t h e Km is v e r y h i g h i n d e e d , since n o e v i d e n c e o f c u r v a t u r e is s e e n o v e r t h e r a n g e e x a m i n e d . T h i s p r o c e s s is r e f e r r e d to as t h e T r k F s y s t e m . N o m u t a n t s a f f e c t i n g it h a v e b e e n i d e n t i f i e d a n d it a p p e a r s to b e p r e s e n t in all s t r a i n s s t u d i e d . T o d e t e r m i n e t h e r o l e o f e a c h o f t h e t h r e e m u t a t i o n s in t h e K - A - D - s t r a i n s , d e r i v a t i v e s o f s u c h a s t r a i n c a r r y i n g o n l y two o f t h e m u t a t i o n s w e r e c o n s t r u c t e d . T h e m o s t d r a m a t i c r e s t o r a t i o n o f t r a n s p o r t o c c u r s w h e n t h e w i l d - t y p e allele at t h e trkA l o c u s is i n t r o d u c e d , r e s u l t i n g in a K - D - s t r a i n . A s s h o w n in F i g . 3, this s t r a i n t a k e s u p K by a p r o c e s s v e r y s i m i l a r to t h a t p r e v i o u s l y n o t e d in w i l d - t y p e strains n o t s u b j e c t e d to K l i m i t a t i o n (Schultz et al., 1963). T h i s is c a l l e d t h e T r k A s y s t e m . F o r t h e e x p e r i m e n t o f Fig. 3 d o n e at 25°C t h e Km was 1.8 m M , a n d V,,ax was 310 ~ m o l / g m i n . I n a n e x p e r i m e n t at 30°C a K i n o f 1.4 m M a n d a V,,ax o f 470 /~mol/g rain w e r e o b t a i n e d . A v e r y h i g h a f f i n i t y s y s t e m f o r K t r a n s p o r t c a l l e d t h e K d p s y s t e m c a n be d e m o n s t r a t e d in A - D - m u t a n t s as well as o t h e r s t r a i n s w h i c h a r e wild t y p e f o r

331

RHOADS, WATERS, AND EPSTEIN K Transport Mutants of E. coli TABLE

II

PROPERTIES OF K UPTAKE SYSTEMS OF E. coli* System

Km

V~ax~

mM

btmol/g min

Kdp T.rkA TrkD TrkF

0.002 1.5 0.5 >500

150 550 40 --

Genetics

Four linked kdp genes Single trkA gene Single (?) trkD gene No mutations known

Other characteristics

Repressible by K Constitutive Constitutive Constitutive (low rate, linearly dependent on K concentration)

* Kinetic data are from this paper; genetic data are from Epstein and Davies, 1970, and from Epstein and Kim, 1971. $ Rates at 37°C for Kdp and TrkA, at 30°C for TrkD. the kdp genes. In A - D - strain TK1004 g r o w n in K5 m e d i u m , K uptake rates were 7.5, 9.1 and 10.8 /xmol/g min at external K concentrations o f 0.01, 0.1, a n d 3 mM, respectively. T h e affinity of this system for K is so high that we were unable to obtain reliable estimates o f the Km f r o m chemical m e a s u r e m e n t s o f K uptake. We therefore used 4ZK in an e x p e r i m e n t in which the cells were allowed to deplete the m e d i u m o f K (Fig. 4). Initial cell K in such experiments is relatively high, a p p r o x i m a t e l y 40 mM in the e x p e r i m e n t o f Fig. 4, because the K d p system allows the cells to scavenge traces o f K in the wash a n d resuspension solutions. I f efflux is negligible the external specific activity will remain constant, a n d radioactivity in the external m e d i u m is directly p r o p o r t i o n a l to K concentration. A double reciprocal plot o f the rate o f K uptake versus K concentration shown in the inset o f Fig. 4 indicated a Km o f 2.5 /xM. A n o t h e r e x p e r i m e n t o f this type yielded a value o f 2 / z M . T h e assumption o f negligible efflux is not critical for these estimates o f the Kin. Efflux will increase external K concentration a n d at the same time r e d u c e the specific activity of external K, two effects which tend to cancel out each other. T h e rate o f u p t a k e by the Kdp system varies over a wide r a n g e d e p e n d i n g on the way the cells are grown. T h e system is not evident at all in cells g r o w n in K115 m e d i u m regardless o f g e n o t y p e , while all strains which are not m u t a n t for the kdp genes express the system at a high rate, r a n g i n g f r o m 140 to 160/xmol/g rain at 37°C after K limitation o f the cells for a p p r o x i m a t e l y 2 h. K limitation in the presence o f c h l o r a m p h e n i c o l does not lead to a p p e a r a n c e o f the system, suggesting that this type o f control is d u e to repression o f synthesis o f one or m o r e protein c o m p o n e n t s o f the K d p system. T h e Kdp system appears to be regulated indirectly by the K c o n c e n t r a t i o n o f the growth m e d i u m to permit the cells to meet their K needs for growth. Growth o f wild-type strains in K5 m e d i u m does not lead to a detectable derepression o f the system as m e a s u r e d by u p t a k e at or below 0.02 mM K where the c o n t r i b u t i o n o f the T r k A a n d T r k D systems (see below) is very small a n d can be estimated. However, as already noted, growth o f A - D - strains in K5 m e d i u m leads to derepression r a n g i n g f r o m 8 to 15/~mol/g rain at 37°C in different e x p e r i m e n t s . These levels are less than 15% o f the m a x i m u m d e r e p r e s s e d levels of the system but not m u c h above the net uptake rate o f 6 ttmol/g min n e e d e d to maintain

332

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 6 zL

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300

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1976

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FIGURE 4 FIGURE 3. Kinetics of K uptake by the T r k A and T r k D systems. Initial rates of uptake were measured after depletion of cell K with DNP. O, K - D - strain T K 1001 at 25°C; the curve drawn is for a saturable process with akin of 1.8 mM and a Vmax of 310 pomol/g rain. 1:3, K - A - strain TK133 at 30°C; the curve represents process with a Km of 0.46 mM and a Vma× of 42 txmol/g rain. FIGURE 4. Measurement of the K,n of uptake by the Kdp system. Wild-type strain FRAG-1 grown at 37°C was subjected to K limitation at 37°C for 90 rain, then depleted of K by DNP treatment, and suspended to a density of 51 /,g/ml in K-free medium containing 2 mg glucose and 50 t*g choramphenicol per milliliter. This suspension was incubated at 21°C for several nlinutes, then at zero time 4ZK (5,000 cpm/nmol) was added to 5.7 p.M. Radioactivity remaining in the m e d i u m was measured in samples obtained by rapid filtration at the times indicated. This measured radioactivity was converted to chemical concentration of K assuming thai specific activity remained constant (see text). External specific activity was calculated by dividing initial radioactivity by the initial external K concentration, the latter obtained indirectly as the change in cell K from control samples before addition of K to the final samples when over 99.5% of the added K had been taken up. T h e inset is a double reciprocal plot of the rate of uptake over each time interval versus the average external K concentration during that time interval. T h e straight line represents a process with a Km of 2.5/xM and a Vm~,.~of 96 ~*mol/g rain. FIGURE

3

physiological K concentrations during exponential growth at 37°C. T h e Kdp system appears to be the only o n e subjected to physiological regulation o f expression by external K since K limitation did not p r o d u c e any detectable changes in the properties o f the T r k A or T r k D systems. T h e t r k D locus affects a m i n o r K transport system not previously detected in wild-type cells since it is o v e r s h a d o w e d by the T r k A system. In K - A - mutants this system o f m o d e s t affinity and rate is seen in relative isolation (Fig. 3). T h e e x p e r i m e n t s h o w n reveals a K m o f 0.46 mM and a Vm~x o f 42 txmol/g min at 30°C. In two other e x p e r i m e n t s , both d o n e at 25°C, the c o r r e s p o n d i n g figures were 0.59 and 0.71 for the Kin, and 19 and 2 4 / , m o l / g rain for the V,,~×.

RrtOADS, WA'rEaS, AND EPSTElY K Transport Mutants of E. coli

333

T h e one K - E - m u t a n t isolated to date has m u c h lower rates o f K u p t a k e t h a n wild-type strains. H o w e v e r , results o f u p t a k e studies were so e x t r e m e l y variable that no clear picture o f the kinetics has e m e r g e d . In six e x p e r i m e n t s at 25°C u p t a k e was saturable; the Km r a n g e d f r o m 0.2 to 2 m M , while estimates o f the V.lax r a n g e d f r o m 20 to 5 0 / z m o l / g rain. T h i s result suggested that such strains may lack u p t a k e by the T r k A system whose function is s o m e h o w affected by the trkE gene p r o d u c t . H o w e v e r , a m u t a n t also lacking the T r k A system, a K - A - E m u t a n t , was c o n s t r u c t e d a n d f o u n d to have u p t a k e characteristics v e r y s i m i l a r to those o f the K - A - D - m u t a n t s (Fig. 2). We t h e r e f o r e conclude that the trkE g e n e p r o d u c t is n e e d e d to obtain high rates o f t r a n s p o r t via both the T r k A a n d the T r k D systems. K u p t a k e in K - B - a n d K - C - strains is rapid a n d similar to that f o u n d in K strains w h e r e u p t a k e is p r i m a r i l y a reflection o f the T r k A system. I n two e x p e r i m e n t s with K - C - strain TK121 at 25°C, the Km was 1.4 a n d 1.6 raM, a n d the Vniax was 245 a n d 230, respectively. T h e results with K - B - strain T K 1 1 0 were similar, with a Km o f 1.2 m M a n d a Vmax o f 500 ~ m o l / g rain at 30°C. T h e s e results, t a k e n with the data o f Fig. 1, indicate that the trkB a n d trkC genes affect K retention, b u t have little or no effect on K u p t a k e . T h e f o u r K u p t a k e systems have b e e n e x a m i n e d in relative isolation; the T r k F system was studied in a m u t a n t lacking the o t h e r three, while each o f the saturable systems was studied in a strain lacking the o t h e r two a n d o v e r a concentration r a n g e w h e r e u p t a k e by the T r k F system is negligible. Is t h e r e an interaction between two systems w h e n both are p r e s e n t , or do they act as i n d e p e n d e n t parallel paths for K t r a n s p o r t ? I f interaction is o c c u r r i n g , the kinetics o f u p t a k e w h e n both are p r e s e n t will not be simply the s u m o f each system as m e a s u r e d alone. T h i s kinetic test for the K d p a n d T r k A systems is shown in Fig. 5. H e r e K u p t a k e was m e a s u r e d in a wild-type strain g r o w n either in K10 m e d i u m to repress the K d p system, or subjected to K starvation to d e r e p r e s s the K d p system. W h e n g r o w n u n d e r r e p r e s s i n g conditions only a single c o m p o n e n t o f u p t a k e with a Km o f 1.2 m M is seen (lower curve), while in the d e r e p r e s s e d cells u p t a k e is nicely described by the u p p e r curve, which is the sum o f the saturable process seen in the r e p r e s s e d cells plus an additional ~:omponent o f u p t a k e constant o v e r the r a n g e o f K concentrations tested. T h i s latter c o m p o n e n t is typical o f the K d p system, a n d the result indicates i n d e p e n d ent f u n c t i o n i n g o f the K d p a n d T r k A systems. In this e x p e r i m e n t net K u p t a k e was p r o d u c e d by osmotic u p s h o c k , a p r o c e d u r e which t e n d e d to give lower a n d s o m e w h a t m o r e variable m a x i m u m rates o f u p t a k e t h a n w h e n K depletion is used to stimulate u p t a k e . Similar results were obtained in two o t h e r e x p e r i m e n t s o f this type, including one in which the K d p system was d e r e p r e s s e d to only 20% o f the extent seen in Fig. 5. We have i g n o r e d the role o f the T r k D system in these e x p e r i m e n t s since that system makes a small c o n t r i b u t i o n c o m p a r e d to the T r k A system. A similar test for the T r k D a n d K d p systems suggests that these systems do interact (Fig. 6). An A - strain was tested with the K d p system, either r e p r e s s e d or partially d e r e p r e s s e d . T h e typical kinetics o f u p t a k e by the T r k D system (lower curve) are c o n v e r t e d a f t e r K d p d e r e p r e s s i o n ( u p p e r curve) to u p t a k e that

334

THE

JOURNAL

OF

GENERAL

A i:

A .l:: 2 4 0 I::

o

I=

5C

PHYSIOLOGY

• VOLUME

67 • 1976

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FIGURE 5

~ (mM)

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K CONCENTRATION(mM)

FIGURE 6

FIGURE 5. Effect of Kdp derepression on K uptake kinetics in a wild-type strain. Strain FRAG-1 was grown at 30°C either in K10 m e d i u m (©), or in K0.05 m e d i u m (El) for approximately 2 h after the time at which K in the medium was exhausted. T h e n the cells were collected and K uptake was measured by the osmotic upshock procedure. T h e lower curve is for a saturable process with a K,, of 1.2 mM and a Vm,x of 145 /zmol/g rain. T h e u p p e r curve is the same curve shifted upwards by a constant 80 ~mol/g min. FIGURE 6. Effect of Kdp derepression in an A strain. Strain TK1005 was grown in Kdp repressing Kl15 medium ( 0 ) or in K0.3 medium ([]) to partially derepress the Kdp system. K uptake was measured at 30°C after K depletion by treatment with DNP. T h e curve is fi)r a saturable process with a K m of 0.3 mM and a Vm~×of 33 ~mol/g min. a p p e a r s to b e i n d e p e n d e n t o f K c o n c e n t r a t i o n , as is t y p i c a l f o r t h e K d p s y s t e m . S i m i l a r r e s u l t s w e r e o b t a i n e d in two o t h e r e x p e r i m e n t s o f this t y p e . D e r e p r e s siGn o f t h e K d p s y s t e m s o m e h o w s u p p r e s s e s all t r a c e s o f a c o m p o n e n t o f u p t a k e with a m o d e r a t e a f f i n i t y f o r K, t y p i c a l o f t h e T r k D s y s t e m . A test f o r a d d i t i v i t y o f t h e T r k F a n d K d p s y s t e m s was p e r f o r m e d by e x a m i n i n g K u p t a k e in t h e r a n g e a b o v e 10 m M in A - D - s t r a i n T K I 0 0 4 a f t e r g r o w t h in K5 m e d i u m to p a r t i a l l y d e r e p r e s s t h e K d p s y s t e m . T h e K d p s y s t e m h a s s u c h a low Km t h a t u p t a k e a b o v e 10 m M s h o u l d b e c o n s t a n t . U p t a k e at 87.5 m M K was 5 . 9 / z m o l / g m i n g r e a t e r t h a n at 12.5 m M K, t h e m e a s u r e m e n t s b e i n g p e r f o r m e d at 30°C. T h i s d i f f e r e n c e is n o t f a r f r o m e x p e c t a t i o n s f o r t h e T r k F s y s t e m , w h o s e r a t e at 37°C i n c r e a s e s f r o m 8 to 11.5 ~ m o l / g m i n o v e r this r a n g e o f K c o n c e n t r a tions (Fig. 2). T e s t s f o r a d d i t i v i t y o f t h e T r k D a n d T r k F s y s t e m s w e r e p e r f o r m e d in t h e s a m e way u s i n g A - K - s t r a i n T K 1 3 3 . C a l c u l a t i o n s f o r t h e T r k D s y s t e m i n d i c a t e t h a t t h e r a t e at 10 m M K is 95% o f t h e m a x i m u m , so t h a t at m o s t a n i n c r e a s e o f s o m e 2 ta,m o l / g m i n is e x p e c t e d w h e n g o i n g f r o m 10 to a n e x t e r n a l K c o n c e n t r a t i o n o f 100 m M . I n t w o e x p e r i m e n t s at 37°C t h e i n c r e a s e f r o m 10 to 100 m M was 6.0 a n d 6.4 ~ m o l / g r a i n , w h i l e in two at 30°C t h e i n c r e a s e was s o m e w h a t l a r g e r , 10.2 a n d 11.7 I z m o l / g m i n . T h e s e v a l u e s a r e in t h e r a n g e e x p e c t e d f o r t h e T r k F s y s t e m , a n d a r e m o r e t h a n c a n b e a t t r i b u t e d to t h e T r k D s y s t e m . K i n e t i c tests f o r i n d e p e n d e n t f u n c t i o n o f t h e T r k D a n d T r k A s y s t e m s p e r f o r m e d at p H 7 a r e n o t s u f f i c i e n t l y sensitive b e c a u s e t h e Km's o f t h e two s y s t e m s are not very different, and the high rate of the TrkA system dominates uptake.

RHOADS, WATERS, AND EPSTEIN K Transport Mutants of E. coli

335

An a t t e m p t to resolve two c o m p o n e n t s was m a d e by m e a s u r i n g u p t a k e at p H 5.5, a condition which stimulates the T r k D system a p p r o x i m a t e l y twofold while inhibiting the T r k A system by a p p r o x i m a t e l y 50% (data not shown). Rates o f K u p t a k e o v e r a r a n g e o f K concentrations f r o m 0.05 to 10 mM in K - strain F R A G 5 were a p p r o x i m a t e l y equal to the s u m o f u p t a k e rates in K - A - strain TK133 a n d K - D - strain T K I 0 0 1 , a result consistent with i n d e p e n d e n t f u n c t i o n i n g o f the T r k A a n d T r k D systems. Double reciprocal plots o f the data are c u r v e d for all three strains, p r e v e n t i n g a simple kinetic resolution o f two c o m p o n e n t s o f u p t a k e in FRAG-5 at low p H . T h e effect o f increasing the g e n e dosage for the kdp a n d trkA genes was tested by c o m p a r i n g u p t a k e o f the c o r r e s p o n d i n g systems in strains partially diploid for the genes with the isogenic haploid strain (Table I I I ) . T h e a m o u n t o f p r o d u c t m a d e f r o m a gene is usually increased in such diploids, the increase r a n g i n g f r o m two- to t h r e e f o l d . For the K d p system the diploid strain t r a n s p o r t s at slightly o v e r twice the rate in the haploid, suggesting that m o r e of the kdp g e n e p r o d u c t s are m a d e in the diploid, a n d that the a m o u n t o f o n e or m o r e o f these p r o d u c t s d e t e r m i n e s the m a x i m u m rate for this system. For the T r k A system the m a x i m u m rate o f u p t a k e in the diploids is not consistently altered, b u t t h e r e seems to be a small reduction in Kin. I f this reduction in Km is d u e to the p r e s e n c e of the e p i s o m e , then the alteration suggests that m o r e trkA gene p r o d u c t is b e i n g m a d e but that the a m o u n t o f this p r o d u c t does not set the m a x i m u m rate for the system. Electroneutrality d u r i n g K u p t a k e can be m a i n t a i n e d by extrusion either o f Na or o f p r o t o n s (Schultz et al., 1963). Cell Na was m e a s u r e d to test w h e t h e r any o f the m u t a n t s d i f f e r e d in the a m o u n t o f Na e x t r u d e d d u r i n g K u p t a k e . Since the cell Na values show m u c h m o r e scatter than the K data (see Methods) only those e x p e r i m e n t s with reasonably small scatter could be used. Most o f the variability is p r o b a b l y d u e to c o n t a m i n a t i o n with m e d i u m Na not c o m p l e t e l y TABLE

III

GENE DOSAGE EFFECTS FOR T H E TrkA AND Kdp SYSTEMS K uptake at 25°C* System

TrkA

Kdp

Strain Km

Vmax

mM

lx,mol/g rain

FRAG-5~: (haploid) FRAG-5/FI41 (diploid)

1.5 1.2

210

160

TK1001 (haploid) TK1001/F141 (diploid)

1.85 1.5

310 270

TK1030 (haploid) TK1030/F100 (diploid)

§ §

71, 77 II 147, 199[1

* K uptake was measured after depletion with DNP. :~ The strain used in the first two experiments is a derivative of FRAG-5 which also has spontaneous ma/A and strA mutations. § Not measured. II Results for two separate experiments are presented.

336

THE

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OF GENERAL

PHYSIOLOGY

• VOLUME

67 • 1976

r e m o v e d by the washing p r o c e d u r e since there were m a n y values m u c h h i g h e r than the majority but n e v e r any that were very m u c h lower. An e x a m p l e of one o f the best e x p e r i m e n t s o f this type is shown in Fig. 7, where the fall in cell Na d u r i n g K u p t a k e by the T r k A system is shown. T h e r e is a linear relationship o f cell Na to cell K, extrusion o f 0.8 equivalents o f Na a c c o m p a n y i n g u p t a k e o f 1 equivalent o f K. Similar e x p e r i m e n t s with strains in which K u p t a k e o c c u r r e d via the K d p system, the T r k D system, the T r k F system, or both the T r k A and T r k D systems yielded ratios r a n g i n g f r o m 0.5 to 1. We d o u b t that any o f these ratios are significantly d i f f e r e n t f r o m each o t h e r since a r a n g e o f 0.5 to 1 was obtained in five e x p e r i m e n t s o f this type with K - E - strain TK142. Since it a p p e a r s that all o f the mutants studied h e r e have very low cell Na when their cell K levels are at or n e a r physiological levels, the a p p a r e n t stoichiometry o f K a n d Na m o v e m e n t s may be d e t e r m i n e d m o r e by the a m o u n t of Na that enters the cell d u r i n g K depletion than by the p r o p e r t i e s o f a particular K t r a n s p o r t system. O u r data d o show that all f o u r K t r a n s p o r t systems are able to take u p K in e x c h a n g e for Na. T h e filter m e a s u r e m e n t s of cell Na suggest that cells not subjected to K depletion have very low concentrations o f Na. E x t r a p o l a t i n g the line o f Fig. 7 to a cell K concentration of 200 m M , which is the physiological K concentration for cells in m e d i u m o f this osmolarity (Epstein and Schultz, 1965), predicts that cell Na will be close to 0. In two e x p e r i m e n t s Na was m e a s u r e d in ceils not subjected to K depletion and s u s p e n d e d in dilute m e d i u m o f 120 mosM a n d an Na concentration o f 58 m M . Cell Na values were 1 +- 3, a n d 10 -+ 4 m M (SE, n = 21 for each), values considerably lower than those r e p o r t e d earlier based on analysis o f cell pellets (Schultz a n d S o l o m o n , 1961). T h e d i f f e r e n c e between filter a n d pellet Na analyses suggests that a considerable a m o u n t o f Na in pellets is loosely b o u n d to the cell envelope a n d is readily r e m o v e d by b r i e f washing. i 15C

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_o ,~ 10C

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FIGURE 7. Relationship of cell Na to {ell K during net K uptake in K-D- strain TKI001. Data are from the same experiment as shown in the top curve of Fig. 3. The point with bars at the upper left represents the mean and standard deviations fi)r six samples of the control suspension before the addition of K. The line, drawn by the method of least squares, has a slope of -0.81.

337

RHOADS, WATERS, AND EPSTEIN K Transport Mutants of E. coli

T h e effect o f the K c o n c e n t r a t i o n o f the m e d i u m on the growth rate o f the m u t a n t s is s h o w n in Fig. 8. T h e results are plotted as the p e r c e n t o f the m a x i m a l growth rate since growth rate o f all o f the d i f f e r e n t m u t a n t s in Kl15 m e d i u m is the same within e x p e r i m e n t a l e r r o r . T h e data o f Fig. 8 is p r e s e n t e d in t e r m s o f initial K c o n c e n t r a t i o n in the m e d i u m . Growth r e d u c e d the values s o m e w h a t , but the c h a n g e was negligible for all but the lowest K concentrations, a n d even below 0.05 m M initial K, the c h a n g e was n e v e r g r e a t e r t h a n 15% d u r i n g the period used to d e t e r m i n e growth rate. T h e logarithmic scale on the abscissa used to allow p r e s e n t a t i o n o f data for all strains in a single figure s o m e w h a t obscures the r a t h e r large differences b e t w e e n strains a n d the steep slopes o f several o f the curves. Using as an index the concentration o f K r e q u i r e d to achieve a halfmaximal growth rate, the m u t a n t s r a n g e f r o m the K - m u t a n t s which require only 0.07 m M to the K - A - D - m u t a n t s which require 10 raM. Hill plots o f the f o u r m u t a n t classes which rise steeply yielded slopes between 2 a n d 3, indicating that the g r o w t h d e p e n d e n c e o f these strains o n e x t e r n a l K can be described as cooperative. G r o w t h rates were m e a s u r e d in the m e d i a listed in T a b l e I V to assess the effect o f p H , osmolarity, a n d o t h e r cations on the K r e q u i r e m e n t s o f the m u t a n t s . For all of the m u t a n t s except the K - E - strain (see below) the curves relating g r o w t h rate to K c o n c e n t r a t i o n in the m e d i a o f T a b l e I V have the same s h a p e as those o f Fig. 8, a n d at high K concentrations all have the s a m e growth rate as that o f a wild-type strain in that m e d i u m . T h u s changes are a d e q u a t e l y described by the K concentrations giving h a l f - m a x i m a l growth rate listed in the table. I n c r e a s i n g the osmolarity or r e d u c i n g p H tends to raise the K r e q u i r e m e n t s o f the m u t a n t s , but the opposite effect is seen in K - A - a n d K - B - strains. T h e effect o f p H on K - A - strains, as c o m p a r e d to K - a n d K - D - strains, is a c c o u n t e d f o r by the different r e s p o n s e o f the T r k A a n d T r k D systems at low p H as n o t e d above. Growth in m a l e a t e - b u f f e r e d m e d i a tested the role o f the s u p p o s e d l y indifferent m a j o r m e d i u m cation on K r e q u i r e m e n t s . In most cases substitution o f e i t h e r Tris or Mg for Na p r o d u c e d no m a j o r effect, a l t h o u g h for several o f the m u t a n t s I00

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