- Email: [email protected]
Growth of the Escherichia coli cell envelope
BIOCHIMIE. 1985, 67, 141-144
Growth of the Escherichia coli cell envelope. Aline JAFFI~ and Richard D'ARI.
hlstitut Jacques Monod, C.N.R.S., Universitd Paris VII, 2 Place Jussieu, 75251 Paris Cedex 05.
R~sum~ - - Le mode de croissance tie l'enveloppe d'Escherichia coli a dtd dtudid par microscopie dlectronique en visualisant la protdine de la membrane externe Lamb marqude spdcifiquement gr6ce h une technique de deux anticorps associds g~ des billes d'or. Une fitsion d'operon pla¢ant le gdne lamB sous contrdle du promoteur de lac permet un arr~t rapide de la synthdse de LamB. Durant la premiere gdndration suivant l'arr~t de cette synth~se nous n'avons pas ddtectd de r~gions ddpourvues de protdine LamB. Les r~sultats sugg~rent fortement que l'ensemble des constituants de la membrane externe n'est pas insdrd dans des zones de croissance prdfdrentielles. Mots-el~s : membrane externe / zone de croissance / LamB.
S u m m a r y - - The growth pattern of the Escherichia coli envelope was studied by hnmunoelectron microscopy, ushlg the outer membrane protehl Lamb specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of Lamb synthesis. In the generation following turn-off 11o lamB-free regions appeared, strongly suggesthlg that bulk outer membrane material is not hlserted in restricted growth zones. Key-words : outer membrane / growth zones / LamB.
Introduction
In bacteria growth of the cell envelope, cell division, DNA replication and genome segregation are important cell cycle events which must be coordinated. The motle of envelope growth may determine the structural changes associated with cell elongation and septation. Jacob, Brenner and Cuzin [1] proposed that envelope growth in Gram negative bacteria takes place in a. central growth zone. Envelope constituents, initially inserted in this zone, would be found further away from it as subsequent growth added new material. If daughter chromosomes were attached to the envelope on opposite sides of the growth zone, proper segregation would be assured. Presumably tl~e growth zone itself would synthesize the septum at the appropriate moment. Each daughter
cell would have to form a new growth zone, and this process would clearly be a key event in the cell cycle and a very early stage of septation. We wished to investigate the potential cell cycle regulation exerted by and on such putative growth zones. We therefore tried to visualize the localisation of new material inserted in the cell envelope. Adam Kepes and his collaborators studied the pattern of insertion o f material into the Escherichia coli envelope by following the segregation of different permeases [2-4]. Insertion patterns have also been studied by direct visualisation of markers in the cytoplasmic membrane [5], the peptidoglycan layer [6-9], the outer membrane [10-13], or a combination o f envelope layers [14, 15], but contradictory conclusions have been drawn, including central growth zones, polar
142
A. Jaffd a n d R. D'Ari
growth zones, random insertion, and mixed modes. Studies with synchronized E. coli cultures indicate that inner menbrane proteins are inserted at an exponentially increasing rate whereas outer membrane proteins and phospholipids are inserted into the cell envelope at a constant rate which doubles abruptly at a particular time in the cell cycle [16-19], compatible with zonal insertion accompanied by the creation of new growth zones at a specific cell age. These observations encouraged us to reinvestigate the growth pattern of the E. coli envelope by following the insertion o f protein into the outer membrane. The method involves specific labelling of an outer membrane protein whose synthesis can be turned on and o f f readily. When its synthesis is turned off, growth zones should a p p e a r as regions o f the bacterial surface devoid o f the marker protein, assuming the latter cannot diffuse rapidly. It is important to realise that such a growth zone would reflect the pattern of insertion, not of the marker protein but of all other outer membrane constituents. Thus if the majority o f outer membrane material is inserted in restricted zones, the growth zones should be detectable, even if a minor fraction is inserted randomly. The outer menbrane marker we chose is the lamB protein, a porin involved in maltose and maltodextrine transport [20,21] and used as receptor by phages L and K10 [22-24]. It can be present at high concentrations in the outer membrane without any deleterious effect on cell growth. Its most important property for our purposes is its tight association with the peptidoglycan [25, 26], suggesting that it cannot diffuse laterally within the outer membrane.
Material and methods The bacterial strain used, provided by Maurice Hofnung, was pop6520, genotype F- thr leu thy lacY tonA supE malTQ. ~j h ° 434), resistant to X vir h 434; the prophage carries the p ~c _ lamB fusion [27]. Cells were grown in broth containing 8 g bactopeptone, 5 g bactotryptone, 5 g NaCI, and 20 mg thiamine per litre and 10-2M MgSO4. -Rabbit antibodies directed against purified L~mB protein were generously provided by Joelle Gabay. -The LamB protein was tagged with anti -- Lamb antibodies and visualised as described by Gabay and Schwartz [26] except that goat antibodies directed against rabbit immunoglobulin and complexed with
20 nm colloidal gold particles purchased from Janssen Pharmaceutica Belgium were used instead of fluoresceine. Ceils were fixed in 1% gluteraldehyde and deposited on Formvar -- Carbon -- coated grids. They were examined in a Philips C410 electron microscope. -Differential rate of l~-galactosidase synthesis was measured as previously described [28].
Results
and
discussion
To be able to turn LamB synthesis on and off rapidly, we took advantage of an operon fusion which places the l a m B gene under control o f the lac promoter [27]. In a l a c Z + strain carrying this pt~C _ l a m B fusion on a ~. prophage, the rate of LamB synthesis was estimated by measuring the differential rate of 13-galactosidase synthesis. Cells cultivated in the presence of 1 0 - : M IPTG (a gratuitous inducer of the lac operon) had a 300to 500- fold higher ~-galactosidase level than uninduced cells, and removal of I P T G by centrifugation resulted in an essentially immediate and drastic drop of the differential rate of synthesis (not shown). The surface location of the Lamb protein was visualized in the electron microscope by a double antibody labelling technique. Cells were first treated with rabbit antibodies directed against purified LamB protein. We next added goat antibodies directed against rabbit immunoglobulin and complexed with 20 nm colloidal gold particles. The E. coli K I 2 strain pop6520, which carries the p taC _ lamB fusion, was grown at 30°C in tryptone broth supplemented with 10 -2 M IPTG. At a density o f 108 cells per ml (after about t5 generations in the presence of IPTG) the bacteria were centrifuged, washed, resuspended in tryptone broth without IPTG, and incubated at 30 °C; doubling time was 45 min. Samples were withdrawn every 10 min, treated with the two antibodies and prepared for electron microscopy as described in Material and Methods. In the 0 rain sample, in which LamB synthesis had been fully induced for 15 generations, all bacteria were covered with a large number of gold particles (135 to > 500) homogeneously distributed over the cell surface (Figure IA). In an uninduced control culture similarly treated, bacteria had 0 to 9 gold particles, reflecting the low level of LamB synthesis in the absence of I P T G and establishing that the labelling technique used was specific to LamB.
Growth of the Escherichia Coli cell envelope
•
143
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B F I G . I.
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Visualisation of LamB protein.
Cells were sampled when fully induced for LamB synthesis (A) and 10 min (B) or 40 min (C) after turn-off of LamB synthesis. Magnification is 40250 X .
In the samples prepared 10, 20, 30 or 40 min after LamB synthesis had been turned off, the distribution of gold particles over the cell surface remained homogeneous (Fig. I B, C and unpublished data). Amongst 50 to 200 bacteria observed per sample, we were unable to detect any cells with regions of low gold particle density, whether at the cell center or at the poles. There was heterogeneity ~in cell size and. in the number of particles per bacterium, which varied over'a 3- to 4-fold range in each sample and decreased with time. Our results strongly suggest that in E. coli outer membrane material is not inserted into the envelope in a restricted growth zone. This is reminiscent of the receht observation that a specific
protein, in fact the LamB protein itself, appeared uniformly throughout the cell surface shortly after induction of its synthesis [12, 13]. Our experimental procedure is straightforward and the results are unambiguous, but the interpretation is not without pitfalls. For example, although the LamB protein is tightly associated with the peptidoglycan in purified sacculi, it is possible that this attachment is looser in vivo and does not prevent lateral diffusion within the outer membrane. Similarly, it is conceivable that the peptidoglycan itself, despite its covalent structure, is subject to constant, rapid rearrangement resulting in randomisation [6, 29]. Also, it should be pointed out that growth zones that are not annular in form (or not perpendicular to the
144
A. Jaffd and R. D',4ri
length axis of the cell) could have escaped detection, since in the electron microscope gold particles in the " t o p " and " b o t t o m " surfaces are superimposed. Our experiments were motivated by the consideration that envelope growth zones might be observable precursors of septa. The possibility that preseptal structures exist is reinforced by the recent discovery o f periseptal annuli [30], annular adhesion zones located on either side o f the septum and bringing together the three envelope layers. This interesting organelle, probably involved in the septation process, may also play a role in preseptal envelope synthesis. Our results suggest that if such a preseptal growth zone exists, it probably governs the insertion of material into the peptidoglycan layer but not into the outer membrane.
Acknowledgements We are deeply gratefid to Joblle Gabay for generously providing anti -- LamB antibodies, Catherine Michon and lrdne Dunia for their patient help with the electron microscopy Rende Tencer for judicious advice. Alaurice Hofnung for the IjJ¢ -- lamB fusion, Richard Schwartzmann for printing the electron micrographs and Florence Haimet for secretarial help. This work was financed hi part b), grants from the Centre National de la Recherche Scientifique bz~ LPO03601 and ASP PIRMED "Antibiotiques").
REFERENCES I. Jacob, F., Brenner, S. & Cuzin, F. (1963) CoM Spring Harbor Syrup. Quant. BioL. 28, 329-348. 2. Autissier, F. & Kepes, A. (1971) Biochimie, 54, 93-101. 3. Autissier, F., Jaffa, A. & Kepes, A. (1971) Mol. Gen. Genet.. 112, 275-288. 4. Kepes, A. & Autissier, F. (1972) Biochim. Biophys. Acta, 265, 443,-469. 5. Donachie, W.D. & Begg, K.J. (1970) Nature, 227, 1220-1224.
6. Ryter, A., Hirota, Y. & Schwarz, U.(1973) J. Mol. BioL, 78, 185-195. 7. Schwarz, U., Ryter, A., Rambach, A., Hellio, R. & Hirota, Y. (1975) J. MoL Biol.. 98, 749-759. 8. Verwer, R.W.H. & Nanninga, N. (1980) J. BacterioL, 144, 327-336. 9. Burman, L.G., Raichler, J. & Park, J.T. (1983) J. BacterioL, 155, 983-988. 10. Ryter, A., Shuman, H. & Schwartz, M. (1975) J. BacterioL, 122, 295-301. II. Begg, K.J. & Donachie, W.D. (1977) J. Bacteriok. 129, 1524-1536. I2. Vos-Scheperkeuter, G.H., Hofnung, M. & Witholt, B. (1984) J. BacterioL, 159, 435-439. 13. Vos-Scheperkeuter, G.H., Pas, E., Brakenhoff, G.J., Nanninga, N. & Witholt, B. (1984) J. BacterioL, 159, 440-447. 14. Beachey, E.H. & Cole, R.M. (1966) .L BacterioL, 92, 1245-1251. 15. Davison, M.T. & Garland, P.B. (1983) Eur..L Biochem.. 130, 589-597. 16. Boyd, A. & Holland, I.B. (1979) Cell, 18, 287-296. 17. Carty, C.E. & Ingrain, L.O. (1981) J. BacterioL, 145, 472-478. 18. Pierucci, O., Melzer, M., Querini, C., Rickert, M. & Krajewski, C. (1981) ,/. BacterioL, 148, 684-696. 19. Joseleau-Petit, D., Kepes, F. & Kepes, A. (1984) Eur. J. Biochem., 139, 605-611. 20. Szmelcman, S. & Hofnung, M. (1975) J. Bacteriol., 124, 112-118. 21. Lucky, M. & Nikaido, H. (1980) Proc. Natl. Acad. Sck USA, 77, 167-171. 22. Randall-Hazelbauer, L. & Schwartz, M. (1973) J'. BacterioL, 116, 1436-1446. 23. Wandersman, C. & Schwartz, M. (1978) Proc. Natl. Acad. ScL USA, 75, 5636-5639. 24. Roa, M. (1979) J. BacterioL, 140, 680-686. 25. Gabay, J. & Yasunaka, K. (1980) Eur. J. Biochem., 104, 13-18. 26. Gabay, J. & Schwartz, M. (1982) J. Biol. Chem., 257, 6627-6630. 27. Hofnung, M., Leponce, E. & Braun-Breton, C. (1981) J. Bacteri6L. 148, 853-860. 28. Casaregola, S., D'Ari, E. & Huisman, O. (1982) Mol. Gen. Genet., 185, 430-439. 29. Goodell, E.W. & Schwar-z, U. (1985) J. Bacteriol, in press. 30. MacAlister, T.J., MacDonald, B. & Rothfield, L.I. (1983) Proc. Natl. Acad. Sci. USA, 80, 1372-1376.
Growth of the Escherichia coli cell envelope. Aline JAFFI~ and Richard D'ARI.
hlstitut Jacques Monod, C.N.R.S., Universitd Paris VII, 2 Place Jussieu, 75251 Paris Cedex 05.
R~sum~ - - Le mode de croissance tie l'enveloppe d'Escherichia coli a dtd dtudid par microscopie dlectronique en visualisant la protdine de la membrane externe Lamb marqude spdcifiquement gr6ce h une technique de deux anticorps associds g~ des billes d'or. Une fitsion d'operon pla¢ant le gdne lamB sous contrdle du promoteur de lac permet un arr~t rapide de la synthdse de LamB. Durant la premiere gdndration suivant l'arr~t de cette synth~se nous n'avons pas ddtectd de r~gions ddpourvues de protdine LamB. Les r~sultats sugg~rent fortement que l'ensemble des constituants de la membrane externe n'est pas insdrd dans des zones de croissance prdfdrentielles. Mots-el~s : membrane externe / zone de croissance / LamB.
S u m m a r y - - The growth pattern of the Escherichia coli envelope was studied by hnmunoelectron microscopy, ushlg the outer membrane protehl Lamb specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of Lamb synthesis. In the generation following turn-off 11o lamB-free regions appeared, strongly suggesthlg that bulk outer membrane material is not hlserted in restricted growth zones. Key-words : outer membrane / growth zones / LamB.
Introduction
In bacteria growth of the cell envelope, cell division, DNA replication and genome segregation are important cell cycle events which must be coordinated. The motle of envelope growth may determine the structural changes associated with cell elongation and septation. Jacob, Brenner and Cuzin [1] proposed that envelope growth in Gram negative bacteria takes place in a. central growth zone. Envelope constituents, initially inserted in this zone, would be found further away from it as subsequent growth added new material. If daughter chromosomes were attached to the envelope on opposite sides of the growth zone, proper segregation would be assured. Presumably tl~e growth zone itself would synthesize the septum at the appropriate moment. Each daughter
cell would have to form a new growth zone, and this process would clearly be a key event in the cell cycle and a very early stage of septation. We wished to investigate the potential cell cycle regulation exerted by and on such putative growth zones. We therefore tried to visualize the localisation of new material inserted in the cell envelope. Adam Kepes and his collaborators studied the pattern of insertion o f material into the Escherichia coli envelope by following the segregation of different permeases [2-4]. Insertion patterns have also been studied by direct visualisation of markers in the cytoplasmic membrane [5], the peptidoglycan layer [6-9], the outer membrane [10-13], or a combination o f envelope layers [14, 15], but contradictory conclusions have been drawn, including central growth zones, polar
142
A. Jaffd a n d R. D'Ari
growth zones, random insertion, and mixed modes. Studies with synchronized E. coli cultures indicate that inner menbrane proteins are inserted at an exponentially increasing rate whereas outer membrane proteins and phospholipids are inserted into the cell envelope at a constant rate which doubles abruptly at a particular time in the cell cycle [16-19], compatible with zonal insertion accompanied by the creation of new growth zones at a specific cell age. These observations encouraged us to reinvestigate the growth pattern of the E. coli envelope by following the insertion o f protein into the outer membrane. The method involves specific labelling of an outer membrane protein whose synthesis can be turned on and o f f readily. When its synthesis is turned off, growth zones should a p p e a r as regions o f the bacterial surface devoid o f the marker protein, assuming the latter cannot diffuse rapidly. It is important to realise that such a growth zone would reflect the pattern of insertion, not of the marker protein but of all other outer membrane constituents. Thus if the majority o f outer membrane material is inserted in restricted zones, the growth zones should be detectable, even if a minor fraction is inserted randomly. The outer menbrane marker we chose is the lamB protein, a porin involved in maltose and maltodextrine transport [20,21] and used as receptor by phages L and K10 [22-24]. It can be present at high concentrations in the outer membrane without any deleterious effect on cell growth. Its most important property for our purposes is its tight association with the peptidoglycan [25, 26], suggesting that it cannot diffuse laterally within the outer membrane.
Material and methods The bacterial strain used, provided by Maurice Hofnung, was pop6520, genotype F- thr leu thy lacY tonA supE malTQ. ~j h ° 434), resistant to X vir h 434; the prophage carries the p ~c _ lamB fusion [27]. Cells were grown in broth containing 8 g bactopeptone, 5 g bactotryptone, 5 g NaCI, and 20 mg thiamine per litre and 10-2M MgSO4. -Rabbit antibodies directed against purified L~mB protein were generously provided by Joelle Gabay. -The LamB protein was tagged with anti -- Lamb antibodies and visualised as described by Gabay and Schwartz [26] except that goat antibodies directed against rabbit immunoglobulin and complexed with
20 nm colloidal gold particles purchased from Janssen Pharmaceutica Belgium were used instead of fluoresceine. Ceils were fixed in 1% gluteraldehyde and deposited on Formvar -- Carbon -- coated grids. They were examined in a Philips C410 electron microscope. -Differential rate of l~-galactosidase synthesis was measured as previously described [28].
Results
and
discussion
To be able to turn LamB synthesis on and off rapidly, we took advantage of an operon fusion which places the l a m B gene under control o f the lac promoter [27]. In a l a c Z + strain carrying this pt~C _ l a m B fusion on a ~. prophage, the rate of LamB synthesis was estimated by measuring the differential rate of 13-galactosidase synthesis. Cells cultivated in the presence of 1 0 - : M IPTG (a gratuitous inducer of the lac operon) had a 300to 500- fold higher ~-galactosidase level than uninduced cells, and removal of I P T G by centrifugation resulted in an essentially immediate and drastic drop of the differential rate of synthesis (not shown). The surface location of the Lamb protein was visualized in the electron microscope by a double antibody labelling technique. Cells were first treated with rabbit antibodies directed against purified LamB protein. We next added goat antibodies directed against rabbit immunoglobulin and complexed with 20 nm colloidal gold particles. The E. coli K I 2 strain pop6520, which carries the p taC _ lamB fusion, was grown at 30°C in tryptone broth supplemented with 10 -2 M IPTG. At a density o f 108 cells per ml (after about t5 generations in the presence of IPTG) the bacteria were centrifuged, washed, resuspended in tryptone broth without IPTG, and incubated at 30 °C; doubling time was 45 min. Samples were withdrawn every 10 min, treated with the two antibodies and prepared for electron microscopy as described in Material and Methods. In the 0 rain sample, in which LamB synthesis had been fully induced for 15 generations, all bacteria were covered with a large number of gold particles (135 to > 500) homogeneously distributed over the cell surface (Figure IA). In an uninduced control culture similarly treated, bacteria had 0 to 9 gold particles, reflecting the low level of LamB synthesis in the absence of I P T G and establishing that the labelling technique used was specific to LamB.
Growth of the Escherichia Coli cell envelope
•
143
2.
.
I
~* "o
**J .~w j
leo
F •
i
:° ~.
o
l•
.:.
•1
** •
•
"'~. %**
,,%
% *
•
"n
%%
-. •o
°°
,
•:
b e
Z
:
.•
;:
. , . °
.•,,. g:.
A
B F I G . I.
-
-
-"
C
Visualisation of LamB protein.
Cells were sampled when fully induced for LamB synthesis (A) and 10 min (B) or 40 min (C) after turn-off of LamB synthesis. Magnification is 40250 X .
In the samples prepared 10, 20, 30 or 40 min after LamB synthesis had been turned off, the distribution of gold particles over the cell surface remained homogeneous (Fig. I B, C and unpublished data). Amongst 50 to 200 bacteria observed per sample, we were unable to detect any cells with regions of low gold particle density, whether at the cell center or at the poles. There was heterogeneity ~in cell size and. in the number of particles per bacterium, which varied over'a 3- to 4-fold range in each sample and decreased with time. Our results strongly suggest that in E. coli outer membrane material is not inserted into the envelope in a restricted growth zone. This is reminiscent of the receht observation that a specific
protein, in fact the LamB protein itself, appeared uniformly throughout the cell surface shortly after induction of its synthesis [12, 13]. Our experimental procedure is straightforward and the results are unambiguous, but the interpretation is not without pitfalls. For example, although the LamB protein is tightly associated with the peptidoglycan in purified sacculi, it is possible that this attachment is looser in vivo and does not prevent lateral diffusion within the outer membrane. Similarly, it is conceivable that the peptidoglycan itself, despite its covalent structure, is subject to constant, rapid rearrangement resulting in randomisation [6, 29]. Also, it should be pointed out that growth zones that are not annular in form (or not perpendicular to the
144
A. Jaffd and R. D',4ri
length axis of the cell) could have escaped detection, since in the electron microscope gold particles in the " t o p " and " b o t t o m " surfaces are superimposed. Our experiments were motivated by the consideration that envelope growth zones might be observable precursors of septa. The possibility that preseptal structures exist is reinforced by the recent discovery o f periseptal annuli [30], annular adhesion zones located on either side o f the septum and bringing together the three envelope layers. This interesting organelle, probably involved in the septation process, may also play a role in preseptal envelope synthesis. Our results suggest that if such a preseptal growth zone exists, it probably governs the insertion of material into the peptidoglycan layer but not into the outer membrane.
Acknowledgements We are deeply gratefid to Joblle Gabay for generously providing anti -- LamB antibodies, Catherine Michon and lrdne Dunia for their patient help with the electron microscopy Rende Tencer for judicious advice. Alaurice Hofnung for the IjJ¢ -- lamB fusion, Richard Schwartzmann for printing the electron micrographs and Florence Haimet for secretarial help. This work was financed hi part b), grants from the Centre National de la Recherche Scientifique bz~ LPO03601 and ASP PIRMED "Antibiotiques").
REFERENCES I. Jacob, F., Brenner, S. & Cuzin, F. (1963) CoM Spring Harbor Syrup. Quant. BioL. 28, 329-348. 2. Autissier, F. & Kepes, A. (1971) Biochimie, 54, 93-101. 3. Autissier, F., Jaffa, A. & Kepes, A. (1971) Mol. Gen. Genet.. 112, 275-288. 4. Kepes, A. & Autissier, F. (1972) Biochim. Biophys. Acta, 265, 443,-469. 5. Donachie, W.D. & Begg, K.J. (1970) Nature, 227, 1220-1224.
6. Ryter, A., Hirota, Y. & Schwarz, U.(1973) J. Mol. BioL, 78, 185-195. 7. Schwarz, U., Ryter, A., Rambach, A., Hellio, R. & Hirota, Y. (1975) J. MoL Biol.. 98, 749-759. 8. Verwer, R.W.H. & Nanninga, N. (1980) J. BacterioL, 144, 327-336. 9. Burman, L.G., Raichler, J. & Park, J.T. (1983) J. BacterioL, 155, 983-988. 10. Ryter, A., Shuman, H. & Schwartz, M. (1975) J. BacterioL, 122, 295-301. II. Begg, K.J. & Donachie, W.D. (1977) J. Bacteriok. 129, 1524-1536. I2. Vos-Scheperkeuter, G.H., Hofnung, M. & Witholt, B. (1984) J. BacterioL, 159, 435-439. 13. Vos-Scheperkeuter, G.H., Pas, E., Brakenhoff, G.J., Nanninga, N. & Witholt, B. (1984) J. BacterioL, 159, 440-447. 14. Beachey, E.H. & Cole, R.M. (1966) .L BacterioL, 92, 1245-1251. 15. Davison, M.T. & Garland, P.B. (1983) Eur..L Biochem.. 130, 589-597. 16. Boyd, A. & Holland, I.B. (1979) Cell, 18, 287-296. 17. Carty, C.E. & Ingrain, L.O. (1981) J. BacterioL, 145, 472-478. 18. Pierucci, O., Melzer, M., Querini, C., Rickert, M. & Krajewski, C. (1981) ,/. BacterioL, 148, 684-696. 19. Joseleau-Petit, D., Kepes, F. & Kepes, A. (1984) Eur. J. Biochem., 139, 605-611. 20. Szmelcman, S. & Hofnung, M. (1975) J. Bacteriol., 124, 112-118. 21. Lucky, M. & Nikaido, H. (1980) Proc. Natl. Acad. Sck USA, 77, 167-171. 22. Randall-Hazelbauer, L. & Schwartz, M. (1973) J'. BacterioL, 116, 1436-1446. 23. Wandersman, C. & Schwartz, M. (1978) Proc. Natl. Acad. ScL USA, 75, 5636-5639. 24. Roa, M. (1979) J. BacterioL, 140, 680-686. 25. Gabay, J. & Yasunaka, K. (1980) Eur. J. Biochem., 104, 13-18. 26. Gabay, J. & Schwartz, M. (1982) J. Biol. Chem., 257, 6627-6630. 27. Hofnung, M., Leponce, E. & Braun-Breton, C. (1981) J. Bacteri6L. 148, 853-860. 28. Casaregola, S., D'Ari, E. & Huisman, O. (1982) Mol. Gen. Genet., 185, 430-439. 29. Goodell, E.W. & Schwar-z, U. (1985) J. Bacteriol, in press. 30. MacAlister, T.J., MacDonald, B. & Rothfield, L.I. (1983) Proc. Natl. Acad. Sci. USA, 80, 1372-1376.