Cell Envelope Proteins Involved in the Transport of Maltose and sn-Glycerol-3-Phosphate in Escherichia coli WINFRIED BOOS Universitiit Konstanz, Fuchbereich Biologie, 7750 Konstanz, Postfach 7 7 3 3 , West Germany

ABSTRACT Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate i n E. coli. The first protein is the receptor for phage A, known to be a n outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the h receptor is to decrease the Km of the transport system without influencing the Vmax of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not i n membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective i n sn-glycerol-3-phosphatetransport.

B The p-methylgalactoside type systems (Boos, '74a) are highly sensitive to It is convenient to classify the active the treatment with the cold osmotic shock transport systems in E. coli in three groups procedure, since they are dependent on according to their sensitivity against os- periplasmic substrate binding proteins motic shock, to the type of proteins in- which are removed from the cell envelope volved, and to the mode of energy coupling during this process. Where genetic data for the accumulation of their substrate. are available it has been found that bindFigure 1 is a schematic representation of ing proteins are essential but not sufficient these systems, exemplified by the lactose, for a functioning transport system. In the the p-methylgalactoside, and the maltose case of the p-methylgalactoside transport systems two additional genes besides that transport system. A The lactose type systems are gen- for the galactose-binding protein are necerally insensitive against the classical cold essary for the complete transport system osmotic shock procedure of Neu and Hep- (Ordal and Adler, '74). It is obvious to pospel ('65) and are still operating in isolated tulate a membrane location for these a s membrane vesicles. These vesicles are yet unknown gene products (Johnson et freed of periplasmic binding proteins, outer al., '75). It is not clear how energy coupling membrane components and cytoplasmic occurs in binding protein dependent syscmstituents. Genetically the best under- tems. Several observations indicate that stood system, that for lactose (Kennedy, Mitchell's protonmotive force is not the '70) consists of only one component. This main source of energy, while phosphatecomponent is tightly membrane bound and bound energy, possibly ATP itself, might the product of the l a c y gene of the lactose play a key role (Berger and Heppel, '74). operon. General agreement exists about Difficulties arise by the observation that the nature of the energy coupling of this entry and exit might occur via different system. As a proton symporter the lactose routes (Parnes and Boos, ' 7 3 ) and exit apcarrier makes use of the membrane poten- pears to be energy dependent (D. B. Wiltial (inside negative) and the pH gradient son, submitted for publication; Ferenci (inside alkaline) across energized mem- and Boos, unpublished observations). It is branes according to the proposal of Mitch- not clear how binding proteins participate ell (see the contribution by Kaback in this in the transport mechanism. One favored idea is to endow them with a Km-factor issue). I. INTRODUCTION

J . CELL.PHYSIOL., 89: 529-542

529

I

I I

I

Fig. 1

V "Shock R e s i s t a n t

f-"N't

~

Protein

M

b

I'

+ mgl A

mgl C

ATP

I I

1

V

4

I

ma1 K

Receptor

ma1 F

"Shock S e n s i t i v e " C y t o p l a s m

V

r

R

I I

Sugar T r a n s p o r t Systems of Active . . . . . . . . . . . . . . . . . . . . . - 2 -- -- -_-_-_---__ --_--E. _c= oP =l io = _-_ -l

I I I 1

I

I

I

I

I

I

I

I

Maltose

Schematic representation to the three different types of active transport systems in Escherzchia coli.

I

I

I

I

I

I

I

I

I

I I I

I

I I

I

I

I I

I I

I

a

I

I

I

0:

I n n e r Membrane

Periplasm

Outer Membrane

R-Methylgalac t o s i d e

I

I

I

Lactose

I

I

I

I

I

I

I

I I

1

I

I

L

I

I

I I

t

$

!

U

1 g

TRANSPORT RELATED ENVELOPE PROTEINS OF E .

like function for a tightly membrane-bound system of low affinity (Robbins and Rotman, '75). Other possibilities have been discussed elsewhere (Silhavy et al., '74). C The maltose-type systems contain in addition to their components i n the cytoplasmic membrane and possibly periplasmic proteins also proteins in the outer membrane. In the case of the maltose transport system it is the A receptor (Szmelcman and Hofnung, '75). Outer transport systems make use of colicin and other phage receptors (Wang and Newton, '71; DiMasi et al., '73; Hantke and Braun, '75).

cou

531

In the following the kinetic parameters of maltose and maltotriose transport in wild-type and A resistant mutants are compared with the binding constants of the maltose-binding protein for these substances. 1 Active transport of maltose Since a non-metabolizable analogue of maltose is not yet available all transport experiments were performed with pop 1000, an amylomaltase negative strain which is unable to utilize maltose or maltotriose as carbon source. Figure 2 shows a typical uptake curve of maltose at 1 p M initial 11. THE MALTOSE TRANSPORT SYSTEM external concentration using 5 x 1 0 7 cells/ AND THE PHAGE A RECEPTOR ml. Under these conditions accumulation Wiesmeyer and Cohn first demonstrated reaches saturation after 6 min. Assuming a transport system which was energy de- a cellular volume of 10-l2 ml (Winkler and pendent and inducible by maltose (Wies- Wilson, '66) one can estimate that the ratio meyer and Cohn, '60). Genetic studies of intra to extracellular radioactivity at the performed later on the maltose system re- equilibrium state of accumulation is i n the vealed that three genes malE, malF, malK order of 8 X 104. However, chromatolocated in the malB region, are essential graphic analysis of the remaining external in the maltose transport capacity (Hofnung, radioactivity revealed that it is no longer '74) and are under the positive control of only maltose but contains in addition a a malT gene. It is clear that this transport compound that runs faster than maltose system is the only in E . coli taking up malt- on paper chromatography. Moreover, the ose, since malE, malF, or malK mutants accumulated radioactivity consists of about do not grow on maltose. A maltose-binding 70% maltose and 30% of the same unprotein was extracted by osmotic shock, known compound which is found in the purified to homogeneity, and shown to be external medium. Despite these unusual a n essential element in maltose transport features the initial uptake of maltose still (Kellerman and Szmelcman, '74), as well gives the kinetic parameters of the transas in maltose chemotaxis (Hazelbauer, '75). port system. Time dependent chromatogThe synthesis of this protein is induced by raphy of the accumulated compounds remaltose and is under the same positive con- veals that this unknown compound is trol as the other proteins of the maltose formed only internally, i.e., not during the system. It is coded for by the malE gene. transport step. The maltose-binding protein is a monomer 2 Kinetic constants for maltose and of 40,000 daltons and it recognizes maltose maltotriose transport via the and the a , 1-+4-linked higher maltodexcomplete transport system trins. From the double reciprocal plots of the Gene lamB, located i n the same operon initial rate of maltose uptake versus difas m a l K , is the structural gene for a n outer membrane protein which acts as a ferent maltose concentrations (fig. 3) one receptor for bacteriophage A (Randall-Ha- obtains a straight line with a n apparent zelbauer and Schwartz, '74). lamB mutants K, of 0.9 Fm and a Vmax of 2.0 nmoles have recently been shown to be impaired per min. per 108 cells. The same plot at in maltose transport when the concentra- higher as well as lower substrate concention of this sugar in the medium is less trations remain linear. This indicates that than 0.1 mM. Furthermore, treatment of maltose uptake is due to a homogeneous wild-type bacteria (lamB+)with antibodies system. Moreover, since the KD of the maltdirected against purified A receptor strong- ose-binding protein for maltose shows the ly reduced the initial rates of maltose same KD as the K m for maltose transport transport, thus demonstrating that the one might conclude that the maltose-binding protein is the only recognition site for phage receptor is a n element of the malt- maltose during the transport step. Howose uptake machinery (Szmelcman and l Szmelcman et al., '76 Hofnung, '75).

I

1

I

1

I

1

I

1

I

I

1

-

/.

/*

0

0 0

-

-

/O

/O

P

-

0

d

I

I

I

I

I

I

1

1

I

I

I

Fig, 2 Uptake of maltose by pop 1000 (malQ7, lamB+). Cells were grown in glycerol containing 50 mM maltose as inducer. Washed cells (5 X lo7 cellslml) and an initial [3H] maltose concentration of 1 p M were used. Plotted are the amounts of maltose accumulated per 2.5 x lO7cells.3 Reprinted from Szmelcman et al. ( ' 7 6 ) with permission of Eur. J. Biochem.. 65: 13-19.

3

--

1.4

v)

0

W

u b

-

0

1.2

X

9 1.0 (u

L

0.8 v 0,

0.6 v)

-0 W

E 0.4 a 1

> , 0.2 0 -0.1

0

I

2

I /MALTOSE Fig. 3

3

4

5

6

7

CONCENTRATION [ I / M ] x

8

10

lo6

Lineweaver-Burk plot of the initial rate of maltose uptake in strain pop 1000 (malQ7,

1am~+).4 4

9

Reprinted from Szmelcman et al. ('76) with permission of Eur. J . Biochem., 65: 13-19.

TRANSPORT RELATED ENVELOPE PROTEINS OF E . COLl

533

s0.3

c

L

0.2

I

u

-I

-0.5 0 0.5 I I/MALTOSE CONCENTRATION [ I / M ] * I O 6

2

Fig. 4 Lineweaver-Burk plot of the initial rate of maltose uptake in strains x , pop 1010 ( m a l Q 7 , lumB+); 0 pop 1 1 0 1 ( m a l Q 7 , l a m B 103).5 5

Reprinted from Szmelcman et al. ( ' 7 6 ) with permission of Eur. J. Biochem., 6 5 : 13-19.

ever, this is not true when the next higher maltodextrin is used as substrate. Double reciprocal plots for the initial rate of maltotriose uptake versus maltotriose concentration (not shown) results again in a straight line with a n apparent K, of 2 ,LM and a Vmax of 1 . 1 nmoles per min. per 108 cells. However, the KD of the maltosebinding protein for maltotriose as measured by fluorescence quenching is 0.15 p M (fig. 6). Yet, despite the discrepancy of Km of transport and KD of binding to the maltose-binding protein the uptake system appears kinetically homogeneous and not mediated via two different recognition sites. Kinetic constants of maltose transport in two mutants defective in the receptor of phage A From strain pop 1000 (malQ7) a mutant was isolated which showed resistance against phage A as well as against host range mutants of A . The mutation was shown to be in l a m B , the structural gene of the receptor protein of phage A . Double reciprocal plots of the initial rate of maltose transport in this mutant against maltose concentrations gave a n apparent K m of 0.5 mM and a V,,, of 0.9 nmoles per min. per 10s cells. Furthermore, a previously 3

characterized missense lam B mutation was introduced in a strain containing the malQ7 (amylomaltase defective) mutation. The determination of K, and Vmax of maltose transport in wild-type and the isogenic 1amB mutant is shown i n figure 4. The mutant exhibited a K, of 0.1 mM i n contrast to the wild-type where the K, was found to be 1 p M . Despite the differences in K, the V,,, for both strains was identical. 'Thus, the absence of a n intact A receptor results in a n apparent increase of the K, for maltose uptake above the Kd of the maltose-binding protein. In the lamB mutants no transport of maltotriose could be measured even at substrate concentrations of 1 mM. This finding is corroborated by the observation that nonsense lam B mutants grow very poorly on maltodextrins (unpublished results). Substrate induced fluorescence quenching of the maltosebinding protein The addition of substrate to the maltosebinding protein drastically alters the intrinsic fluorescence properties of this protein. Figure 5B shows the emission spectrum (excitation at 280 nm) and figure 5 A the excitation spectrum (emission at 350 nm) of the maltose-binding protein in the 4

534

WINFRIED BOOS

24

Cl 20 W

V

Z

2

16

W

[I:

0

3 I2

LL W

1

G-I 8 W

[r

4

0

I

260

I

L

280

I

I

300

300

320

340

360

300

320

340

360

WAVELENGTH ( n m ) Fig. 5 Fluorescence excitation and emission spectra of the maltose-binding protein. In this and all subsequent figures the excitation and emission slits were 7.5 nm, the temperature was 22", and the buffer was buffer A, pH 7 . 1 . The dotted lines show the relative fluorescence of the protein (3.5 pg/ml) alone, while the solid lines show fluorescence in the presence of 0.1 mM maltose. A, excitation spectra with an emission wavelength of 350 nm; B, emission spectra with an excitation wavelength of 280 n m , C, emission spectra with excitation wavelength of 290 nm (upper curves), (1) and 295 nm (lower curves) (2).6 6

Reprinted from Szmelcman et al. ('76) with permission of Eur. J . Blochem. 65: 13-19

presence and absence of saturating concentrations of maltose (0.1 mM). Excitation maximum occurs at 281 nm and emission maximum at 348 nm. The addition of maltose results in quenching of the fluorescence emission with a maximal effect at an emission wavelength of 333 nm. The emission spectra remain unchanged in relative shape when the protein is excited at 280, 285, or 290 nm (fig. 5C); indicating that emission is due to mainly one type of chromophore, probably tryptophan (Udenfriend, '62). Addition of maltose not only results in an emission quenching but also in a slight but reproducible shift of 2 n m to longer wavelength. The fluorescence quenching of the maltose-binding protein is specific: the sugars glucose, a-methylglucoside, lactose as well as isomaltose in 1 mM concentrations have no effect and do not inhibit the fluorescence change caused by maltose. On the other hand higher maltodextrins (maltotriose, maltotetraose, maltopentaose, maltoheptaose, and cyclic heptaose) which have previously been shown to interfere with binding of maltose to the maltose-binding protein (Kellerman and Szmelcman, '74) exhibit fluo-

rescence quenching of the maltose-binding protein that is in the same order of magnitude as that produced by maltose.

Titratwn of the fluorescence quenching effect of the maltose-binding protezn b y maltose and maltotriose Figure 6 shows the dependence of fluorescence quenching of the maltose-binding protein on the total concentration of maltose. Maximum quenching occurs at concentration above 10 p M with a half maximal response at 1 pM. Similar experiments performed with maltotriose gave a maximal response above concentration of 2 p M with a half maximal response at 0.15 p M (data not shown). Therefore, maltosebinding protein has a higher affinity for maltotriose than for maltose. However, the affinity for longer maltodextrins such as maltotetraose, pentaose, or heptaose is the same as for maltotriose. By comparing the kinetic parameters of transport in vivo with the binding parameters of the maltose-binding protein in vitro the following points can be made: Reciprocal plots of the initial rate of uptake versus substrate concentration 5

TRANSPORT RELATED ENVELOPE PROTEINS OF E .

24

coLr

535

I

0

0

0

g -20 I

rO/

0

Z 3

16

0 W 0 W 0

12

cn

W

E 8 3 J

IL

$

4

0

I

I o-8

Io

-~

I

I

I I I I

I

o-6

L

Io

-~

Io

-~

Io

-~

MALTOSE C O N C E N T R A T I O N ( M 1 Fig. 6 Concentration dependence of maltose induced fluorescence quenching. Maltose was added to a protein containing cuvette in 5 p1 aliquots and the fluorescence emission was measured at 350 nm, excitation was at 280 nm. The same volume of buffer was added to an identical control cuvette and the percent quenching was corrected for the observed dilution. Maltose-binding protein concentration was -0 -, 7 pglml; - 0-,14 pg/ml.7 7

Reprinted from Szmelcman et al. ('76) with permission of Eur. J . Biochem., 6 5 : 13-19

with all substrates and mutants were linear over large concentration ranges. Thus, there is no evidence for the independent functioning of two recognition sites; A receptor must therefore function in sequence with the maltose-binding protein rather than in parallel with it. With maltose as substrate the apparent Km of transport into the wild-type and KD of binding to the binding protein was identical, 1 CLM.Thus, in the presence of A receptor the access of maltose to the maltose-binding protein is not limited. Mutations in the receptor for phage A increased the Km for maltose transport by a factor of 100-500 without altering the maximal rate of transport at saturating substrate concentrations. Since A receptor is known to be located in the outer membrane (Randall-Hazelbauer and Schwartz, '73) this result suggests that in absence of this protein dif-

fusion of maltose through the outer membrane is the rate limiting step in transport at low concentrations of this substrate. With maltotriose as a substrate the K m of transport (2 pM) in the wild-type is thirteen fold higher than the Kd of binding to the binding protein. This suggests that even in the presence of h receptor diffusion of maltotriose through the outer membrane is the limiting step at low concentration of substrate. The two lamB strains tested do not transport maltotriose. Therefore, in the absence of A receptor the outer membrane is essentially impermeable to trisaccharides, as suggested by others (Nakae, '75; Nakae and Nikaido, '75). The very bulky substrate cyclic heptaose is bound to the binding protein with a KD that is in the same order of magnitude as maltotriose but it cannot be transported via the maltose transport

536

WINFRIED BOOS

system, not even in the wild-type, nor does it inhibit maltose transport, presumably because it cannot pass the outer membrane barrier. How does the A receptor facilitate the permeability through the outer membrane for maltose and higher maltodextrins? Purified A receptor did not bind maltose after isolation with cholate (unpublished results). Until now attempts to detect binding activity of the A receptor either in vitro or in vivo using malE mutants defective in maltose-binding protein were equally negative. Under the experimental conditions a binding affinity with a K D of 5 p M and lower would have been detected. In analogy to Inouye's model regarding the formation of pores by lipoprotein in the outer membrane (Inouye, '74) it is tempting to speculate that the A receptor protein also plays the role of a "pore" for maltose and higher maltodextrins. According to such a model the A receptor may have no binding affinity for its substrates. One may speculate that the passage of other molecules, particularly of carbohydrates of similar size as maltose or smaller, would equally well be facilitated by the A receptor. Evidence that this is so would require the availability of a system in which the rate of transport is limited by diffusion across the outer membrane. Other transport systems have recently been reported to require, in addition to their intrinsic transport proteins, outer membrane components otherwise recognized to be phage or colicin receptors (Wang and Newton, '71; DiMasi et al., '73; Hantke and Braun, '75). Whether the role of these proteins is similar to that proposed here for the A receptor is not clear at present. Quite interestingly the maltose-binding protein exhibits a higher affinity for maltotriose and other maltodextrins than for maltose. This suggests that the binding site was selected to recognize maltotriose rather than maltose. Taken together with the existence of the lamB protein, which is more stringently needed for growth on maltodextrins than on maltose, this observation suggests that the maltose system in fact evolved to utilize the mixture of linear oligosaccharides originating from the hydrolysis of starch and glycogen. 111. A PERIPLASMIC PROTEIN RELATED TO THE sn-GLYCEROL-3-PHOSPHATE TRANSPORT S Y S T E M OF E S C H E R I C H I A COLI

*

The sn-glycerol-3-phosphate (GLP) transport system in E. coli appears to be ideally suited for a combined genetic-biochemical analysis. There exists a n easy selection

procedure for transport negative @ l p T ) strains by selecting for resistance against phosphonomycin (Hendlin et al., '69, Lin, '70, Venkateswasan and Wu, '72). Transport positive revertants can be obtained by growth on GLP. Also, constitutive @lpR) strains can be selected (Lin et al., '62). glpT strains have been found 50% cotransducible by phage P1 to the nalA marker and the map position of 43 min. on the E . coli chromosome has been proposed for the glpT mutation (Cozzarelli et al., '68; Kistler and Lin, '71). From these studies it cannot be determined whether the gZpT gene conslists of one or more cistrons. Unlike most transport systems in E. coli the GLP transport system is homogeneous, i.e., the substrate is transported by only one system. This system has been characterized as a n energy dependent active transport system (Km 12 p M ) translocating the substrate without chemical alteration (Hayashi et al., '64). Despite these advantages no attempts have been made to study the system with biochemical methods. In particular, it is not clear whether the system is mediated via a periplasmic binding protein (Boos, '74b) or a typical membrane bound system active in membrane vesicles of the Kabackosome type (Kaback, '72). In the following we describe the identification of a periplasmic protein that is related to the sn-glycerol-3-phosphate transport system. 1

Two dimensional gel electrophoresis of shock fluid from E. coli

Figure 7 shows the pattern of a Comassie Blue stained two dimensional polyacrylamide gel slab of the periplasmic proteins obtained by osmotic shock from strain LA3400 (wild-type) after growth with 0 . 4 % glycerol as carbon source. Separation of the proteins occurs in the first dimension (left to right) in 8 M urea predominantly according to their electrical charge. The second dimension (top to bottom) occurs in SDS emphasizing differences in molecular weight. With this tlechnique nearly all proteins in the osmotic shock fluid of E. coli can be separated. Therefore, if the synthesis of a yet unknown protein belonging to this compartment can be influenced by induction, repression, or mutation it should easily be recognixed by this analytical technique. Based on this rationale we observed a protein among the periplasmic proteins 2

Silhavy et al.,'76

TRANSPORT RELATED ENVELOPE PROTEINS OF E

COLI

537

Fig. 7 Two dimensional polyacrylamide gel electrophoresis of shock fluid of strain LA 3400 (wild-type) grown i n the presence of glycerol. First dimension is electrophoresis in 8 M urea, PH 8.4 followed by electrophoresis in 0.2% SDS, p H 6.48 as described (Johnson et al., '75). About 300 p g protein were applied. The numbers and molecular weights correspond to the following proteins: 1, GLPT; 2, galactose-binding protein (Boos and Gordon, '71); 3, ribosebinding protein (Willis and Furlong, '741, 4 , maltose-binding protein (Kellerman a n d Szmelcm a n , '74).R 8

Reprinted from Silhavy et al. ('76) with permission of J. Bacteriology, 126: 951-958

that appeared to be inducible during growth in the presence of glycerol.

The sn-Glycerol-3-Phosphate transport protein (GLPT) Figures 8 A and B show the detailed area of the two dimensional gels from shock fluids of strain LA-3400 grown with trehalose and glycerol as carbon source. One protein spot (1) appears inducible by glycerol. The protein exhibits under the denaturing conditions of the first dimension an unusual high negative charge. In the second dimension (SDS) a n approximate molecular weight of 40,000 daltons can be estimated. The same analysis of the shock protein of a glycerol constitutive glpR strain (fig. 8C) grown in the presence of succinate demonstrates that the protein in question is under the control of the glpR regulatory gene. In addition, as seen i n figure 8D growth of the glpR strain under anaerobic conditions in the presence of 2

glycerol and fumarate (Freedberg and Lin, '73) GLPT appears fully induced. From the availability of this protein in the periplasmic shock fluid it seemed likely that it might be either the gene product of the glpF (glycerol facilitator) (Richey and Lin, '72) or the glpT gene, known to be involved in GLP transport (Cozzarelli et al., '68). glpT Mutant analysis By growth in the presence of phosphonomycin an inhibitor of cell wall synthesis and substrate of the GLP transport system we isolated spontaneous glpT mutants (Venkateswasan and Wu, '72). The two dimensional polyacrylamide gel electrophoresis of the shock proteins or two independently isolated glpT strains, derivatives of the constitutive glpR strain 72 are shown in figures 9A and C. Both mutant preparations miss the protein entirely. Simultaneously both strains fail to grow on 3

538

WINFRIED BOOS

Fig. 8 Detailed pictures of two dimensional polyacrylamide gel slabs of shock fluids from several strains: A, LA 3400 (wild-type) grown on trehalose; B, LA 3400 grown on glycerol; C, strain 72 ( g l p R ) grown on succinate; D, strain 72 grown on glycerol i n the presence of fumarate under anaerobic conditions. Experimental conditions as i n figure 7.9 9

Reprinted from Silhavy et al. ( ' 7 6 ) with permission of J . Bacteriology, 126: 951-958

GLP but grow normally on glycerol. Their duce GLPT. Figurses 9 B and D show that defect must therefore be confined i n the one strain (LA 3406) has fully regained transport of GLP. Measurements of GLP the capability to prloduce GLPT while strain uptake in these mutants in comparison to LA 3407 shows only a partial reversion. its wild-type parent is shown in figure 10. The extent by which GLPT reappears in Using a substrate concentration of 0.3 p M , these revertants is reflected in their transfar below 12 pM, the Km of the system port activity of GLP (fig. 10B). (Hayashi et al., '64), the mutants exhibit Shock fluids of several other mutants less than 5 % transport activity of their that fail to grow on GLP after isolation for parent. Transport of GLP in the wild-type resistance against phosphonomycin were is competitively inhibited by orthophos- analyzed by the two dimensional gel elecphate with a Ki of 20 mM (not shown). This trophoresis (not shown). Some of these muvalue agrees well with the Ki determined tants (LA 3405) exhibit a reduced amount previously (Hayashi et al., '64) and indeed of GLPT, others (LA 3404) show no apparcharacterizes the measured GLP uptake ent alteration nejther i n the amount of as being mediated by the GLP transport GLPT nor in its position on the gel slab. system. Application of the cold osmotic This is consistent with the observation in shock procedure of Neu and Heppel ('65) several binding protein dependent transto the wild-type strains clearly shows that port systems: the periplasmic component the GLP transport system is dependent on is essential but no't sufficient for transport a periplasmic component (fig. 1OA). Spon- activity (Boos, '74b). taneous revertants of the two mutants described above to grow again on GLP were 4 Genetic analysis of the isoZated glpT mutants isolated and measured for the transport activity as well as their capability to proSince phosphonomycin resistant mutants

TRANSPORT RELATED ENVELOPE PROTEINS OF E. COLI

539

Fig. 9 Detailed pictures of two dimensional polyacrylamide gel slabs of shock fluids from the following g l p T mutants and their revertants. A, LA 3401 ( g l p T ) grown on glycerol; B, LA 3406 ( g l p T + revertant of LA 3401) grown o n glycerol; C, LA 3402 ( g l p T ) grown on glycerol; D, LA 3407 ( g l p T + revertant of LA 3402) grown on glycerol. The number 1 designates GLPT. Experimental conditions as in figure 7.10 10

Reprinted from Silhavy et al. ('761 with permission of J . Bacteriology, 126: 951-958.

nave previously been reported to arise also from mutation of the hexosephosphate transport system as well as in the phosphoenolpyruvate uridine diphospho-N-acetylglucosamine enolpyruvyl transferase (Venkateswasan and Wu, '72) it was necessary to genetically characterize our GLP transport mutants. A lysate of phage P1 grown on a nalA strain was introduced into two GLP transport negative strains selecting for growth on GLP. The transductants were scored for nalA. In the first transduction with strain LA 3403 of 42 transductants 18 or 43% were nalA. In the second transduction with strain LA 3401 of 79 transductants 49 or 62% were nalA. These results are similar to the published cotransduction frequency of 50 % between g l p T and nalA (Cozzarelli et al., '68; Kistler and E n , '71) and establishes the mutation of these strains as g l p T . The shock fluids of four g l p T + transductions were checked by two

dimensional electrophoresis and found to contain GLPT (not shown). From the analogy of a series of other transport systems that are mediated via periplasmic components (Boos, '74b) it seems likely that GLPT is a binding protein specific for GLP. Yet, all attempts to measure in crude shock fluids binding activity for GLP even with the most sensitive method of ultrafiltration (Paulus, '70) have failed so far to give positive results. However, with a n expected binding affinity of 12 p M (the Km of GLP transport in whole cells (Hayashi et al., '64)) and the relative small amount of GLPT in crude shock fluids, binding activity might be difficult to detect. Another complication arises from the observation that all shock fluids independent of their content of GLPT contain a n enzymatic activity that splits GLP in glycerol and Pi. This is surprising in view of the fact that all strains used in this study are p h o A and therefore do not produce the

WINFRIED BOOS

B

A

/

A

t

0

0

&-------

---$-I

20

40

60

80

/'

40

20

60

80

Time [ s e c ] Fig. 10 GLP transport activities of wild-type mutant a n d revertant strains. The bacterial cultures were resuspended i n growth medium minus carbon source at a n optical density of 0.5 (576 nm). ['*C] GLP was added at a n initial concentration of 0.3 pM. The data are expressed i n amount GLP taken u p per 0.5 ml samples. All operations were done at room tema n d after (0-0-0) osmotic shock (1); B, muperature. A, strain LA 3400 before (0-0-0) tant strain LA 3401 (+.-.), revertant LA 3406 ( 0 - 0 - 0 ) ;mutant strain LA 3402 (A-A-A), revertant strain LA 3407 ( A - A - A ) . l l I r

Reprinted from Silhavy et al. ( ' 7 6 ) with permission of J . Bacteriology, 126: 951-958

periplasmic alkaline phosphatase. By paper chromatographic analysis it can be seen that under conditions similar to those used for the binding assay by ultrafiltration 3 0 4 0 % of the GLP is hydrolyzed i n one hour. Therefore, a meaningful test for binding activity or any enzymatic activity of GLPT has to await the purification of GLPT. Since transport of GLP appears to depend on the presence of the periplasmic GLPT one would not expect that membrane vesicles of the Kabackosome-type are able to transport GLP. However, stimulation of membrane-bound transport systems by GLP as well as its own transport has been reported (Barnes and Kaback, '71; Heppel et al., '72). Measurement of possible residual amounts of GLPT in mem-

brane vesicles might explain this discrepancy. Alternatively periplasmic transport components might be essential for transport in whole cells but not in membrane vesicles, a phenomenon reported for the E. coli dicarboxylic transport system (Lo and Sanwal, '75). LITERATURE CITED Barnes, E. M., and H . R. Kaback 1971 Mechanism of active transport i n isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydrogcmase a n d P-galactoside transport i n Escherichia coli membrane vesicles. J. Biol. Chem., 246: !55185522. Berger, E. A., and L.. A . Heppel 1974 Different mechanisms of energy coupling for the shocksensitive and shock-resistant amino acids permease of Escherichia coli. J . Biol. Chem., 249: 7 74 7-7755.

TRANSPORT RELATED ENVELOPE PROTEINS OF E. Boos, W. 1974a Pro and contra transport carriers; the role of the galactose-binding protein in the p-methylgalactoside transport system of Escherichia coli. Current Topics i n Membranes and Transport, 5: 51-136. 1974b Bacterial transport. Ann. Rev. Biochem., 43: 123-146. Boos, W., and A. S. Gordon 1971 Transport properties of the galactose-binding protein of Escherichia coli. Occurrence of two conformational states. J. Biol. Chem., 246: 621-628. Cozzarelli, N. R., W. B. Freedberg and E. C. C. Lin 1968 Genetic control of the La-glycerolphosphate system in E. coli. J. Mol. Biol., 31: 371487. DiMasi, D. R., J. C. White, C. A. Schnaitman and C. Bradbeer 1973 Transport of vitamin BI2 in Escherichia coli: Common receptor sites for vitamin BZZ and the E colicins on the outer membrane of the cell envelope. J. Bacteriol., 115: 5 0 6 5 1 3 . Freedberg, W. B., and E. C. C. Lin 1973 Three kinds of controls affecting the expression of the glp regulon i n Escherichia coli. J. Bacteriol., 115: 816-823. Hantke, K., and V. Braun 1975 Membrane receptor dependent iron transport i n Escherichia coli. FEBS Letters, 49: 3 0 1 3 0 5 . Hayashi, S., J. P. Koch and E. C. C. Lin 1964 Active transport of La-glycerophosphate in Escherichia coli. J. Biol. Chem., 239: 30983105. Hazelbauer, G. L. 1975 Maltose chemoreceptor of Escherichia coli. J. Bacteriol., 122: 206-214. Hendlin, D., E. 0. Stapley, M. Jackson, H. Wallick, A. K. Miller, F. J. Wolf, T . W. Miller, L. Chaiet, F. M. Kahan, E. L. Foltz and H. B. Woodruff 1969 Phosphonomycin, a new antibiotic produced by strains of streptomyces. Science, 166: 122-123. Heppel, L. A., B. P. Rosen, J. Friedberg, E. A. Berger and J. H. Weiner 1972 Studies on binding proteins, periplasmic enzymes and active transport in Escherichia coli. In: The Molecular Basis of Biological Transport. J. F. Woessner, Jr., and F. Huijing, eds. Academic Press, New York and London, pp. 133-150. Hofnung, M. 1974 Divergent operons and the genetic structure of the maltose B region i n Escherichia coli K-12. Genetics, 76: 169-184. Inouye, M. 1974 A three-dimensional molecular assembly model of a lipoprotein from the Escherichia coli outer membrane. Proc. Natl. Acad. Sci. USA, 71 : 2396-2400. Johnson, W. C., T. J. Silhavy and W. Boos 1975 Two-dimensional polyacrylamide gel electrophoresis of envelope proteins of Escherichia coli. Applied Microbiol., 29: 4 0 5 4 1 3 . Kaback, H. R. 1972 Transport across isolated bacterial cytoplasmic membrane. Biochim. Biophys. Acta, 265: 3 6 7 4 1 6 . Kellerman, O., and S. Szmelcman 1974 Active transport of maltose in Escherichia coli K12. Involvement of a “periplasmic” maltose-binding protein. Eur. J. Biochem., 47: 139-149. Kennedy, E. P. 1970 The lactose permease system of Escherichia coli. In: The Lactose Operon. J. R. Beckwith and D. Zipser, eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 49-92. Kistler, W. S., and E. C. C. Lin 1971 Anaerobic La-glycerophosphate dehydrogenase of Escherichia coli: Its genetic locus and its physiological role. J. Bacteriol., 108: 1224-1234. Lin, E. C. C. 1970 The genetics of bacterial transport systems. Ann. Rev. Genetics, 4: 225262. Lin, E. C . C., J. P. Koch, T. M. Chused and S. E. Jorgensen 1962 Utilization of La-glycerophosphate by Escherichia coli without hydroly-

cou

54 1

sis. Proc. Natl. Acad. Sci. USA, 48: 2145-2150. Lo, T. C. Y., and B. D. Sanwal 1975 Isolation of the soluble substrate recognition component of the dicarboxylate transport system of Escherichia coli. J. Biol. Chem., 250: 1600-1602. Nakae, T. 1975 Outer membrane of Salmonella typhimurium: Reconstitution of sucrose-permeable membrane vesicles. Biochem. Biophys. Res. Commun., 64: 1224-1230. Nakae, T., and H. Nikaido 1975 Outer membrane as a diffusion barrier in Salmonella t y p h i muriurn. J. Biol. Chem., 250: 7359-7365. Neu, H. C., and L. A. Heppel 1965 The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem., 240: 3685-3692. Ordal, G. W., and J. Adler 1974 Isolation and complementation of mutants i n galactose taxis and transport. J. Bacteriol., 11 7: 5 0 9 4 1 6 . Parnes, J. R., and W. Boos 1973 Energy coupling of the p-methylgalactoside transport system of Escherichia coli. J. Biol. Chem., 248: 44364445. Paulus, H. 1970 A rapid and sensitive method for measuring the binding of radioactive ligands to protein. Anal. Biochem., 32: 91-100. Randall-Hazelbauer, L., and M. Schwartz 1973 Isolation of the bacteriophage lambda receptor from Escherichia coli. J. Bacteriol., 116: 14361446. Richey, D. P., and E. C. C. Lin 1972 Importance of facilitated diffusion for effective utilization of glycerol by Escherichiu coli. J . Bacteriol., 112: 784-790. Rabbins, A. R., and B. Rotman 1975 Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12. Proc. Natl. Acad. Sci. USA, 72: 4 2 3 4 2 7 . Silhavy, T. J., W. Boos and H. M. Kalckar 1974 The role of the Escherichia coli galactose-binding protein in galactose transport and chemotaxis. In: 25th Mosbacher Colloquium, Biochemistry of Sensory Functions. L. Jaenicke, ed. Springer Verlag, Berlin. Silhavy, T. J., I. Hartig-Beecken and W. Boos 1976 A periplasmic protein related to the snglycerol-3-phosphate transport system of Escherichia coli. J. Bacteriol., 126: 951-958. Szmelcman, S.,and M. Hofnung 1975 Maltose transport in Escherichia coli K12. Involvement of the bacteriophage lambda receptor. J. Bacteriol., 124: 112-118. Szmelcman, S., M. Schwartz, T. J. Silhavy and W. Boos 1976 Maltose transport in Escherichia coli K12. Eur. J. Biochem., 65: 13-19. Udenfriend, S. 1962 Fluorescence Assay. In: Biology and Medicine. Academic Press, New York. Venkateswasan, P. S., and H. C. P. Wu 1972 Isolation and characterization of a phosphonomycin resistant mutant of Escherichia coli K12. J. Bacteriol., 110: 935-944. Wang, C. C., and A. Newton 1971 An additional step in the transport of iron defined by the tonB of Escherichia coli. J. Biol. Chem., 246: 2 14 7-2 15 1 . Wiesmeyer, H., and M. Cohn 1960 The characterization of the pathway of maltose utilization by Escherichia coli. 111. A description of the concentrating mechanism. Biochim. Biophys. Acta, 39: 4 4 0 4 4 7 . Willis, R. C., and C. E. Furlong 1974 Purification and properties of a ribose-binding protein. J. Biol. Chem., 249: 6926-6929. Winkler, H. H., and T. H. Wilson 1966 The role of energy coupling in the transport of p-galactosides by Escherichia coli. J. Biol. Chem., 241: 2200-221 1.

Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli.

Cell Envelope Proteins Involved in the Transport of Maltose and sn-Glycerol-3-Phosphate in Escherichia coli WINFRIED BOOS Universitiit Konstanz, Fuchb...
1003KB Sizes 0 Downloads 0 Views