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Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement
J. Mol.
Biol.
(1991) 217, 429-439
Individual Domains of Colicins Confer Specificity in Colicin Uptake, in Pore-properties and in Immunity Requirement H. Benedetti’,
M. Frenette’Jf, D. Baty’, M. Knibiehler’ F. Pattus’ and C. Lazdunski’
‘Centre de Biochimie et de Biologic Mole’culaire de C.N.R.S. 31 Chemin Joseph Aiguier, B.P. 71 13402 Marseille Cedex 9, France 2European Molecular Biology Laboratory Postfach 10.2209, 6900 Heidelberg, Germany (Received 22 May
1990; accepted 17 September 1990)
Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and El. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin El is located in the N-terminal domain of colicin El; (4) the 183 N-terminal amino acid residues of colicin El are sufficient to promote ElAA uptake and thus probably colicin El uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin El by their respective immunity proteins.
1. Introduction The protein antibiotics colicin A and colicin El kill sensitive Escherichia coli by forming voltagedependent ion channels (Pattus et al., 1983a,b; Cramer et al., 1983; Davidson et al., 1984) thereby destroying the cell’s energy potential. They belong to the major group of colicins, which comprises colicins A, B, El, Ia, Ib, K and N, and are of particular interest to membrane biologists because they are unusual in being water-soluble transmembrane proteins. The vitamin B,, receptor, BtuB, is used by colicins A and El as a receptor protein at the surface of target cells, but colicin A also needs the OmpF porin for this step (Cavard & Lazdunski, 1981; Sabet & Schnaitmann, 1971). After binding to their receptors, colicins A and El translocate across the cell envelope to reach the cytoplasmic membrane and may interact with other membrane proteins during this step. Many mutations in the to1 locus (Nagel de Zwaig & Luria, 1967; Nomura & Witten, 1967; Davies & Reeves, 1975; Sun & Webster, 1987) render cells tolerant to t Present address: Dkpartement de Biochimie, Facultk des Sciences et de GEnie, Universitb Laval, Foy, Q&bee GlK 7P4, Canada.
Ste
these colicins and resistant to infection by filamentous bacteriophages (fl, Ml3 or fd), probably by blocking the transfer of colicins and DNA across the envelope. Colicin A requires TolA, TolB, TolQ and TolR with a low requirement for TolC (related to TolC effect on OmpF synthesis), while colicin El absolutely requires TolC, TolA and TolQ but neither TolB nor TolR (Nagel de Zwaig & Luria, 1967; Nomura & Witten, 1967). In addition, results reported by Benedetti et al. (1989) suggest that OmpF and BtuB play a part in colicin A and colicin El translocation, respectively. Thus, while both colicins use BtuB as a receptor, their uptake mechanism differs. It has been suggested that the amino-terminal domain of colicin A and of colicin El are involved in translocation (Ohno-Iwashita & Imahori, 1982; Martinez et al., 1983; Baty et al., 1988). One might therefore ask whether the specific features in the uptake of these two pore-forming colieins are related to their respective amino-terminal domains. The pore properties of colicin El and A have been extensively investigated using planar lipid bilayers and liposomes (Pattus et al., 1982, 1983a,b; Davidson et al., 1984, 1985). These properties differ with regard to extent significant to a
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H. Benedetti
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pH-dependence of conductance and kinetics of closing after a voltage step. It would be of interest to investigate whether these properties are intrinsic properties of the C-terminal pore-forming domains. Finally, direct evidence for specific recognition between the C-terminal domain of colicin A and its corresponding immunity protein has never been provided. Colicin A immunity protein, designated Cai, is a 178 residue protein (Lloubes et al., 1984; Geli et al., 1986) with four transmembrane-spanning regions (Geli et al., 1989); it may interact with the pore-forming domain of cohcin A. To address these questions, six hybrid colicins containing various combinations of colicin A and colicin El domains were constructed (Frenette et al., 1990, accompanying paper). The hybrid colicins are designated by a three-letter code, in which the first letter corresponds to the N-terminal domain, the second to the central domain and the third to the C-terminal domain. For example, AElA means N-terminal domain of colicin A, central domain of colicin El and C-terminal domain of colicin A. The properties of these hybrids are presented here. The results demonstrate that dependence on TolB, TolR and OmpF for colicin A and TolC for colicin El is associated with their N-terminal domain. Furthermore, the immunity proteins only need the C-terminal domain of the corresponding colicin to specifically protect against their lethal action. Some hybrids turned out to have a very weak activity in viva while the activity in vitro was comparable to that of colicin A or El; thus, uptake of these hybrids appears to be affected and interactions between domains seem important for this step. As shown by assays in planar lipid bilayers, interactions between domains also affect the characteristics of the pore of colicin A. These interactions become even more critical when pore-forming colicins with different uptake systems (TonB and (H. Benedetti, Tol-dependent) are recombined unpublished result).
2. Materials and Methods (a) Bacterial strains and growth conditions The E. co& MC4100 strain was used as a host for the various colicinogenic plasmids to produce eolicins A, El and hybrid colicins for subsequent purifications. E. coli MC4100 strain (wild-type, OmpF- or BtuB(Cavard & Lazdunski, 1981)), E. co& K12 C600 strain (TolB- A593 (Sun & Webster, 1987) and TolC- ~602 (Davies & Reeves, 1975)) and E. coli GM1 strain (TPS 300 (orf 3 :: Cm) containing the toER mutation (Sun & Webster, 1987)) were used for the activity tests. They were grown in LB or M9 medium supplemented with thiamine (1 pg/pl) and 94 y0 (v/v) glycerol (for the fluorescence assay). MC4100 pColA9 and MC4100 pColE1 strains, immune to colicin A and El, respectively, were also used to test the activity of hybrid colicins. (b) Activity
ccssays
(i) Plate assay Dilutions from 10-l to 10-a of a 1 mg/ml stock solution of colicin A, coliein El and hybrid colicins were assayed
et al. by spotting lo-p1 portions on freshly seeded lawns of the MC4100 strain. The highest dilution for which a clear zone is observed on the MC4100 lawn is indicated in Table I. Activity of the hybrids has been assayed on lawns of the OmpF-, TolB-, TolC- and TolR- cells and cells carrying immunity to colicin A and El by spotting 10 ~1 of the stock solution.
(ii) Liquid culture assay To calculate the number of killing units, the SDS assay (Cavard & Lazdunski, 1979) was used on E. eoli strain MC4100. Cells of E. coli MC4100 were grown in LB medium at 37°C to A 6oo = 92. A volume of 100 yl of eolicin (A, El or hybrid) diluted in sodium phosphate buffer (10 mM-NaH,PO,/Na,HPO,, pH 6.8) containing @l% (v/v) Triton X-100 was added to 1.25 ml of this culture and incubated for 20 min at 37 “C with shaking, As a control, sodium phosphate was used instead of a colicin dilution. Subsequently, 100 ~1 of SDS (7.5 mg/ml) was added and turbidity at 600 nm was measured after 10 mm shaking at 37°C. The percentage of surviving cells was estimated from the turbidity ratio of the coliein-treated and the cont,rol samples. The number of killing units for each hybrid was calculated as described (Cavard &
Lazdunski, 1979). To test activity on OmpF- and BtuB- MC4100 ceils under normal conditions and to compare it to activity in bypass conditions, the routine assay was used. Cells of E. co& MC4100 were grown in LB medium to -4,,, = 02. A volume (10 id) of a colicin dilution (or sodium phosphate buffer as a control) was added to 100~1 of this culture and incubated for 20 min at 37°C with shaking. Subsequently, 1.25 ml of LB medium was added and the absorbance at 600 nm of the culture monitored until that of the control reached 0.4. The same number of killing units on MC4100 wild-type cells were obtained with this method a,nd the previous one. (iii) Assays in bypass conditions These tests were performed as described (Cavard $ Lazdunski, 1981). Conditions are the same as above except that the culture of E. coli MC4100 (wild-type. OmpF- or BtuB-) was grown to AeoO = 0.8, washed 3 times in IO mM-sodium phosphate buffer and diluted itat. this buffer to &,,e = 62. (iv) Fluorescence assays The bacteria were grown to 8 x IO* cells/ml in MM9 medium. The concentration of 8-anilinol-naphthalene sulphonic acid, magnesium salt (ANS) added to the cell suspension was %7x 10m5 w As described by Cramer & Phillips (1970), the excitation wavelength was 360 nm and the fluorescence emission was near 480 nm. After addition of AK’S, different amounts of colicins or hybrid colicins were added in a 1 ml cuvette (the volume added did not exceed 1.7% of the total) and fluorescence increase was measured as a function of time. The initial rate of fluorescence increase (which reflects membrane depolarization rate) was measured as a function of the amount of colicin or hybrid added in the cuvette. (c) Poye-forming
activity
in lipid planar
bilayers
Phosphatidylcholine from soybeans (Sigma Chemicai Co., St Louis, MO, type TIS) was purified according to Kagawa & Racker (1971). Bilayers were formed across a 150 pm hole pretreated with a 1 : 40 solution of hexadeeane in pentane from 1 mg/ml solutions of the phospholipid as described by Schlinder (1980).
Speci$c
Properties
of Individual
The membrane voltage was clamped to the desired value using 2 Ag/AgCl electrodes connected to the buffer in the 2 compartments via agar bridges. The membrane current was amplified using an I-V converter with an operational amplifier (Burr Brown 3528) and feedback resistors ranging from lo7 to lo9 R. The truns compartment was connected to the I-V converter and held at virtual ground potential. The sign of the membrane potential refers to the cis side of the membrane. Current was defined as positive when cations flowed into the tram compartment. Protein was always added to the cis side while the aqueous solution was stirred vigorously with a magnetic bar.
Domains in Colicins
431
Table 1 of hybrid colicins
Activity Colicins
Dilutionst
A
K.U./mg x lo-“1
lo4 lo4 5 2 2 lo4 lo3 lo4
El AElEl AElA AAEl ElAA ElAEl ElElA
8 15
N.D. N.D. N.D. 5 1.5 8
t Highest dilution (of a stock solution 1 mg/ml) for which a (d) Purification
of hybrid
colicins
The synthesis of colicins and hybrids was induced by adding mitomycin C to 300 rig/ml to growing cultures at A 6oo = 1.0. After 5 h of induction, the cultures were centrifuged at 10,OOOg for 15 min. For wild-type colicin A, mutated colicin A (encoded by pColA9 SX, see the accompanying paper) and AElA hybrid, supernatants of the centrifuged cultures were precipitated with 55% saturated ammonium sulphate overnight. The precipitate was centrifuged at 10,OOOg for 1 h. The pellet was resuspended and dialysed against 2 x 4 1 of 10 mw-phosphate buffer (Nap,), pH 68, 1 mM-EDTA. The dialysed material was applied on an S-Fastflow anionic exchanger (Pharmacia) in the same buffer, and proteins were eluted with a 0 M to @4 iv-Nacl gradient. Samples of the fractions were analysed by SDS/ polyacrylamide gel electrophoresis and immunoblotting, the fractions containing material reacting with anticolicin A monoclonal antibodies were pooled and dialysed against 25 mM-Mes (pH 55), 1 mM-EDTA. The dialysate was applied to a Mono S column (Pharmacia) and the proteins were eluted with a 0 M to @4 M-NaCl gradient. The fractions were analysed as desc@bed above. Fractions showing a &gle band on SDS/polyacrylamide gel electrophoresis Coomassie blue-stained gels that reacted with anti-colicin A monoclonal antibodies in immunoblotting were pooled, freeze-dried and stored at -20°C. For wild-type, mutated colicin El (encoded by pBRE1 XX, see the accompanying paper) and ElAEl hybrid, the centrifuged cells were extracted with 1 M-NaCl according to the method of Schwartz & Helinski (1971). The salt extract was dialysed against 2 x 4 1 of 10 mnrNaPi buffer (pH 7), 1 mM-EDTA and applied to a Mono Q column (Pharmacia) in the same buffer. The excluded fraction was dialysed against 50 miw-sodium borate (pH %5), 1 mm-EDTA; the dialysate was applied to a Mono S column (Pharmacia) and proteins were eluted with a 0 M to @4 M-NaCl gradient. Fractions showing a single band on SDS/polyacrylamide gel electrophoresis Coomassie blue-stained gels that reacted with anti-colicin El antibodies in immunoblotting were pooled, freezedried and stored at -20%. Hybrids ElAA, ElElA, AElEl and AAEl, which were not released from producing cells to the extracellular medium, were purified as follows. Centrifuged cells were sonicated in 10 mm-phosphate buffer (pH 6%; Nap,) containing 1 mM-EDTA (a metal protease inhibitor), 12 miw-N-dodecyl-fl, N-dimethyl-amino300 mM-NaCl, a-propane sulphonate (sulfobeta’ine SBl2), @02 mMphenylmethylsulphonyl fluoride (a serine protease inhibitor). The extract was centrifuged at 20,000 g for 20 min and the supernatant was dialysed against 2 x 4 1 of 10 mMNaP, buffer (pH 7) containing 1 mM-EDTA, 6 mi+-SBI2. The dialysate was applied to an S-Fastflow anionic
clear zone is observed on a lawn of MC1400 cells.
$ Comparison of the number of killing units (K.U.)/mg of colicins A, El and the most active hybrid colicins (tested as described in Materials and Methods). N.D. Not done.
exchange (Pharmacia) column in the Same buffer. Proteins were eluted with a 0 M to @4 M-NaCl gradient. Analysis of the fractions and the 2nd part of the purifications involving a chromatography on Mono S column were performed as described for colicin A and AElA but with the addition of 3 mM-SBl2 to each buffer.
3. Results (a) Activity
of hybrid
colicins
Purified colicins and hybrids were assayed on the sensitive indicator strain MC4100 for activity as described in Material and Methods. The results are presented in Table 1. The Ser/Val substitution at position 168 in colicin A and the Thr/Leu substitution at position 184 in colicin El, as well as the Arg/Lys substitutions at positions 394 and 379 in colicin A and colicin El, respectively, did not alter activity,
expression
or release
of mutated
colicins
(see the accompanying paper). Out of the six hybrids, all were active but to varying degrees. The activity of hybrids ElAA and ElElA was the same as that of the colicins A or El, where it was ten times lower with the hybrid ElAEl. But hybrids AElEl, AElA and AAEI were about lo4 times less active than colicins A and El (Table 1). The fluorescence assay enabled us to evaluate the rate of depolarization with hybrids and to compare these rates to those observed with colicins A and El. A typical example is shown in Figure 1. The initial rate of fluorescence increase (which reflects membrane depolarization rate) was measured as a function of colicin and hybrid concentrations and the dose-dependent rate was plotted (Fig. 2). Colicin A caused very fast membrane depolarization, while depolarization was much slower with colicin El. In addition, there was a marked difference between the two colicins with regard to the dose dependence of the rate. Using 1 pg of colicin A per cuvette, we obtained the same rate of depolarization as with 9 pg of colicin El per cuvette. For hybrids, behaviour in the kinetics of depolarization seemed to follow that of the C-terminal domain. Hybrids
H. Benedetti
432
eL al.
0.8 ? x 6 0.6 :: 6 2 0.4 .-5 z 0.2 z
0
5
IO
15
Colvins
20
25
(pg)
Figure 2. Variation of the initial rate of fluorescence increase as a function of the amount of colicin added; (o)A, (A) El, (m) ElAA. (0) ElElA, (0) ElAEl, (A) AElEl. The rate of fluorescence increase is expressed in relative
fluorescence/s.
r 0
1000
500 Time (5) (b)
Figure 1. Effect of colicin B or hybrid colicins on the fluorescence of APU‘S bound to E. coli MC4100 cells. Relative fluorescence at 480 nm is plotted as a function of time after the addition of colicin or hybrid colicins as described in Material and Methods. (a) Addition of colicin A: a, @05 pg; b, @I ,ug; c, 05 pg; d, 1 pg. (b) Addition of hybrid coiicins: a, 5pg of ElAEl; b, 0.5pg of ElAA; c, 5 pg of ElElA.
ElAA and ElElA caused fast depolarization at low concentration, like colicin A, while ElAEl caused slow depolarization even at high concentration. No information could be obtained from hybrids AElEl, AElA and AAEl because their low activity resulted in too weak an increase of fluorescence. The fact that every hybrid was active demonstrated that the translocation domain and the receptor-binding domain of colicins A and El could be recombined in a functional way, though with varying degrees of efficiency. We have observed that even though OmpF and BtuB act as receptor proteins for colicin A, they do not play exactly the same role; OmpF is involved both as a receptor and in translocation across the outer membrane, whereas BtuB is involved only at the receptor binding step (Cavard & Lazdunski, 1981; Benedetti et aZ., 1989). Tn contrast, for colicin El, OmpF is not involved either as a receptor or in translocation, while BtuB is involved in both processes. It was therefore of interest to assay the activity of hybrids on OmpF mutant cells in order to investigate which domain in colicin A conferred OmpF dependence. This was checked by plate and liquid cult’ure assays. The results are presented in Table 2. MC4100 OmpFstrain is still sensitive to colicin El at a dilution as high as 10-4 of a stock solution at a concentration of 1 mg/ml. As AElEl, AElA and AAEl hybrids are 10e4 times less active than colicin El, the 1 mg/ml stock solution of these hybrids should exhibit killing spots on OmpF- cells if this stra.in was sensitive. No killing spot was
observed, therefore the active hybrids AElEl, AElA and AAEl, which contained the N-terminal domain of coiicin A, were OmpF-dependent. This dependence was confined to the 168 N-terminal residues of colicin A since it was not observed with hybrids ElAA and ElAEl. TolB and TolR proteins are necessary for coliein A uptake but not for colicin El. In contrast, TolC protein is absolutely required for colicin El entry, whereas it is only partially needed for colicin A: probably because to02 caused a marked decrease in OmpF synthesis (Misra & Reeves 19873. Again, to investigate which domain was responsible for TolB, TolR, and TolC dependence, the activity of all hybrids was tested on TolB-, TolRand TolCstrains (Table 2). TolB- and TolR- cells were as sensitive to colicin El as wild-type MC4100 cells; thus; if they were sensitive to AElA, AAEl and BElEl hybrids, killing spots should have been observed. They were, on the other hand, fully sensitive to ElAA, EIEIA and ElAEl hybrids. These results clearly indicated
Activity of co&ins A and El and hybrid colicins ox ,rnutant cells (resistant or tolerant) and immune cells Activity
on difkrent
types of cells-i Cells immune to$
Colicins A El AElEl AElA AAEl EIAA ElElA ElAEl
OmpF + z +
ToIF + + + +
TolR-
ToiC-
Colicin A
Colicin El
t t f +
+ + + + -
+ + + +
i + + + -
i- + , Active; -. inactive. 1 The indicator strains were MC4100 pColAY and MC4100 pColE1 for assaying immunity to colicin A and El, respectively.
Specific
Properties
of Individual
100
100
in Colicins
433
-
80
ao-
60
60
40
40
20
20
0
Domains
0 I00
IO’
to*
103
IO4
100
105
10’
IO’
103
104
Id5
(6)
I00
2 a0 ;
80
60
60
z2 40
40
v) 20
20 0
100
IO’
IO”
I03
(b)
IO4
IO5
IO6
(e)
100
100
a0
a0
60
60
40
40
20
20
0 IO0
IO’
102
103
0 I04
105
100
IO’
I02
IO3
IO4
I05
Dilutions (cl
(f 1
Figure 3. Activity assays of hybrid colicins under normal and bypass conditions. The activity on MC4100 cells (wildtype, OmpF- and BtuB-) was tested in bypass and normal conditions as described in Materials and Methods. In each graph is indicated the colicin or hybrid tested in bold letters, and at the right the symbols for each curve. BC designates wild-type cells, BP bypass conditions, B- BtuB- cells and O- OmpF- cells. (a) AAEl activity assays under normal and bypass conditions. (b) Colicin A activity assays under normal and bypass conditions. (c) AElEl activity assays under normal and bypass conditions. (d) ElElA activity assays under normal and bypass conditions. (e) Colicin El activity assays under normal and bypass conditions. (f) ElAA activity assays under normal and bypass conditions. that TolB and TolR dependence was strictly related to the N-terminal domain of colicin A. TolC- cells were lo2 times less sensitive to colicin A than the MC4100 wild-type cells. Accordingly, the highest dilut)ion of colicin A stock solution (1 mg/ml) able to exhibit killing spots was 10m2. This suggested that 1 mg/ml solutions of AAEl , AEl A and AElEl (lo4 times less active than colicin A) should not produce any spot on a TolC- lawn even if they were active. In fact, they exhibited very turbid killing spots with a large diameter. The same turbid aspect was observed with dilutions of colicin A higher t,han 10’. As expected, colicin El stock solution (1 mg/ml) exhibited no spot and the sa’me results were observed with ElAA and ElElA (as active as colicin A or colicin El on wild-type cells) and ElAEl (10 times less active than colicin A or colicin El on wild-type cells). These results (Table 2) suggested that the N-terminal domain of colicin El is responsible for TolC dependence.
(b) Activity
of hybrid colicins shock conditions
under osmotic
It has been reported that resistant cells could be killed by colicins under osmotic shock conditions
(Cavard & Lazdunski, 1981; Tilby et al., 1978; Bishop et al., 1985; Benedetti et al., 1989). This technique allows BtuB receptor bypassing for colicin A, although limitation due to translocation (Tol-dependent step) across the envelope remains (Bourdineaud et al., 1989). In these conditions, the lethal action of colicin A is also enhanced. In contrast, the same conditions decrease the lethal activity of colicin El and BtuB cannot be bypassed. It was of interest to test the activity of the various hybrids in these conditions to determine whether one structural domain or more, and which were involved in these various features. ElElA and ElAEl behaved exactly like colicin El (Fig. 3(e) and (f)). There was a marked difference in sensitivity (lo-fold difference in dilution) between BtuB+ and BtuBcells under bypass conditions and both were OmpF independent because OmpFcells were as sensitive as wild-type cells under normal and bypass conditions. Furthermore, as observed with colicin El (Benedetti et al., 1989), the hybrids were less active on wild-type cells after the bypass treatment. As the only colicin El domain common to these two hybrids is the N-terminal domain, it is tempting to assign to this domain the features of colicin El in bypass conditions. However, the behaviour of the
434
H. Benedetti et al.
Table 3 Characteristics of colicins and hybrid colicins in bypass experiments Enhanced activity
No requirement for BtuB
Lower activity
Requirement for BtuB
Colicin A AAEl AElEl ElAA
Colicin A AAEl
Colicin El ElElA ElAEl
Colicin El AElEl AElA EIAA ElElA ElAEl
ElAA hybrid was not consistent with this hypothesis. As expected, the ElAA hybrid was OmpF independent. There was a marked difference in sensitivity (IO-fold difference in dilution) between BtuB+ and BtuB- cells in bypass conditions (Fig. 3(d)) but, like colicin A, it was more active on wild-type cells in bypass conditions than in normal conditions. Hybrids possessing the N-terminal domain of colicin A, AElEl, AElA and AAEl were lo4 times less active than colicin A in normal conditions. This is why (since in this assay colicin solution was diluted IO-fold by the cells) such a high survival percentage was observed for the wild-type cells in normal conditions. All these hybrids were more active on wild-type cells under bypass conditions than under normal conditions, like colicin A and ElAA hybrid. Furthermore, OmpF- cells were much less sensitive than wild-type cells under bypass conditions (Fig. 3(a) to (c)). Like colicin A, AAEl was as active on BtuBcells as on wild-type cells under bypass conditions (Fig. 3(a)). In contrast, BtuB seemed important for AElA and AElEl translocation, because cells deficient in this protein were less sensitive to the lethal action of the proteins than wild-type cells in bypass conditions. These results are summarized in Table 3. It clearly appears that the presence of both the
N-terminal and central domains of eolicin A is required for the bypass of BtuB protein under low ionic strength conditions. These two domains are then fully responsible for this colicin A charaeteristic. However, no precise domain could be held responsible for either a lower (like colicin El) OF higher-(like colicin A) activity on wild-type cells in bypass conditions compared to normal conditions. (c) Pore-forming ability of hybrids in lipid planar bilayers Extensive studies of channel properties of eolicin A and El have been carried out (see Slatin, 1988 and references therein). These studies demonstrated the existence of two different forms of channel with colicin A depending on pH. In contrast, only one form was observed with the C-t.erminal thermolytic fragment of colicin A. The ion channel properties of colicin El and its C-terminal fragment are more difficult to distinguish. In this study, we did not try to differentiate between colicin El and colicin El C-terminal domain type of channel properties. The hybrid colicins were thus classified into three categories according to their ion channel properties in lipid planar bilayers: eolicin A, colicin A C-terminal and colicin El classes. The major difference between the colicin El class and the two colicin A classes is the affinity for lipids at neutral pH. At pH 7.2, 94 ng of colicin A was sufficient to induce a large conductance increase when injected in front of lipid of planar bilayers (Fig. 4). In contrast, appearanee ion channel events occurred only when more than 1 pg of colicin El was injected under the sa,me conditions (Figs 6(b) and (c) and 7(a) to (e)). At neutral pH, a voltage step from 100 mV to 40 mV or negative potentials induces closing of colicin A channels (Fig. 5(b)). At the same ~33, the channels formed by the colicin A C-terminal peptide do not close at 40 mV and close slowly at negative potentials (Fig. 7(d)). The colicin El channels do not close at these potentials at neutral pH but close 70 mV
80 mv
90 mV 45 mV
(a) Figure 4. Pore-forming activity of the eoliein A type. Stepwise current increase after injection of @4 ng of (a) EPAA hybrid protein or (b) colicin A in front of an asolectin planar bilayer. The 2 proteins behave identically. The channels close after a voltage step to 45 mV or 70 mV (50 mw-Hepes (pH 7.2), 1 m-NH&L, 5 mmCaCl,). Broken lines indicate the zero-current level.
Xpeci$c Properties of Individual
Domains in Colicins
435
90 mV
PL_ 40 mV
(b)
(0)
Figure 5. Macroscopic conductances. The current relaxation kinetics after a voltage step from 90 mV to 40 mV are identical for (a) hybrid ElAA and (b) colicin A. Same conditions as in Fig. 4.
_---L. m
u _.____ _--_-_ _.__.. -._-_ m (cl 4_._.__ I min
Figure 6. Single channel events observed with different hybrids. (a) Hybrid AElA, 94 ng injected. This hybrid belongs to the colicin A C-terminal type of channel. The major single channel conductance is 40( +5) pS. (b) and (c) Hybrids ElAEl and AAEl, respectively; 2 pg injected. These hybrids belong to the colicin El class. The predominant single channel conductance is 30( k5) pS (50 mm-Hepes (pH 7.2) I M-NH&L, 5 mM-Call,). Broken lines indiea.te the zero-current level.
H. Benedetti
436
et al.
9q
90mVI
90~ -90
mV
-90
-90
mV
(a)
(b)
-90
mV
Cc)
mV
Cd)
Figure 7. Current traces in response to a voltage step. (a) and (c) AAEl, 10 pg injected. (b) AElEl, 10 pg injected. (d) AEIA, 10 ng injected. (c) and (d) 50 mM-Hepes (pH 7.2), 1 M-KH$L, 5 mM-Cd&. (a) and (b) 50 m&r-Tris-acetate (pH 5.0); 1 M-NH,CL, 5 mM-CaCl,. (a) and (c): note that AAEl conductance does not inactivate at pH 7.2 but inact’ivates quickly at pH 5.0 after a voltage step to - 90 mV. AAEl and AEl El belong to the colicin El type of channel. In contrast to colicin A and ElAA (Fig. 5), the hybrid AElA conductance inactivates slowly at -90 mV at neutral pH. This hybrid belongs to the colicin A C-terminal type of channel. Broken lines indicat,e the zero-current level. at negative potentials at acidic pH (Fig. 7(a) and (c)). Although the single channel conductances of colicin A and colicin El in NH,Cl at pH 7.2 differ significantly (40( f 5) pS and 25( 2 5) pS), they were not chosen to classify the hybrids due to the presence of sublevels of conductance, which render quantitative analysis rather difficult. Qualitative analysis of the hybrid protein conductances did not change the classification shown in Table 4. All the hybrid mutants, even those with low activity in wivo (AElEl, AElA and AAEI) were able to form ion channels in lipid planar bilayers with the same efficiency as the representatives of their categories. Among the hybrid mutants, ElAA is the only one that could not be distinguished from colicin A (Figs 4 and 5). The other mutants containing the C-terminal domain of colicin A (ElElA and AElA) formed channels similar to those formed by the colicin A C-terminal peptide (Figs 6(a) and 7(d)). These results strongly favour the hypothesis presented previously (Frenette et al., 1989) that the receptor-binding domain of colicin A may interact
with t,he pore-forming domain at neutral pHj affecting the channel gating by membrane potent’ial. As expected, the hybrids containing the poreforming doma,in of coliein El displayed low affinity for membranes at neutral pH and the same gating properties as colicin El (Table 4, Fig. 7(a) to (e)).
(d) The iwmunity in colicin A and colicin E:I :‘s spec$cally directed again& the C-terminal domabn It has been reported with the pore-forming coliein Ta and Ib that immunity is specifically directed against the C-terminal domain (Mankovich et al.. 1984). However, this had never been demonstrated with colicin A. Here, we observed that ElElA was inactive against cells producing immunity to eolicin A while, as expected, they were sensitive to AE7 El, AAEl and E1AEl. This result unambiguously demonstrates that immunity to colicin A (Cai protein) is directed against the C-t,erminaI domain of colicin A. A similar conclusion applied to colicin El as the hybrid AAEl was inactive against cells producing immunity to colicin El.
Table 4 BehaGour Sffinity membranes
Colicin
pH7
A El Th A$ AElEl SElA $AEl ElElA ElAA ElAEl
High Low High Low High Low High High Low
of hybrid
for
colicins
Inactivation
in planar at pH 7i
at +40
mv
++ ++ -
i -, So inactivation; + + , slow inactivation; + Th A, thermolysin fragment of colicin A.
-80
bilayers
lipid
Inactivation at pH 5t
IIll-
+++ ++ ++ ++ +++ + + + fast inactivation
- 90 mv
++ ++ ++ +++ ++ +++ ++ ++ +++
Type of pore Colicin Colicin Th A Colicin Th A Colicin Th A4 Colicin Colicin
A El El El B El
Xpecijk Properties of Individual
4. Discussion Colicins A and El share some homology in their C-terminal domain (Lazdunski et al., 1988). However, they differ to a large extent with regard to the sequence of the domains involved in receptor binding and translocation. Colicin El, indeed, requires TolC protein (an outer membrane protein) but not TolB, TolR nor OmpF for its action. In contrast, colicin A requires TolB, TolR, OmpF but not TolC (the low dependence probably reflects the effect of toZC on OmpF synthesis). It has been reported that colicin D (a protein synthesis inhibitor) that shares receptor specificity (Fep A) and TonB dependence for translocation with the pore-forming colicin B has an N-terminal and central domain very similar to that of this colicin. This result demonstrated that receptor and translocation domains for a pore-forming colicin are functional also for a colicin that has a cytoplasmic target (Roos et al., 1989). This suggested that the assembly of colicin genes occurred during evolution through recombination of DNA fragments encoding domains responsible for colicin activity and uptake. In this study along similar lines, we attempted to construct hybrid colicins derived from colicin A and colicin E 1. It was possible that by exchanging the N-terminal domains of colicin A and El, the requirements for TolB, TolR, TolC and OmpF could be exchanged. We also wanted to check whether the receptor-binding domain from a given colicin could be combined with the translocation domain of another colicin in a functional way, allowing penetration of the toxin into the cell. It might provide definite evidence as to whether there was specific recognition between the C-terminal domain of colicin A and colicin El and their immunity proteins, as with colicin Ia and Ib experimental et al., 1984). This (Mankovich approach assumed that domains were folded independently and that strong interactions were not required for a functional uptake. Six different constructs were made (Table 1). The six hybrids were found to be active on sensitive cells. Three of them, AEIEl, AElA and AAEl, exhibited very low activity in vivo. However, they were as active in vitro as the original colicins A or El constituting their C-terminal domain. This suggested that the uptake for these hybrids was a limiting step. It is striking that these three hybrids have the N-terminal domain of colicin A and use OmpF for translocation. These features may be compa.red to those of secretory proteins. In this case, it has been demonstrated that their conformation is a constraint on the routing and translocation of proteins. Extensive studies on different combinations of signal peptides and secretory proteins have strongly suggested that not all combinations are compatible (Pugsley, 1989). Therefore, with colicins there is a possibility that each amino-terminal domain has specifically evolved to be most compatible with the two other colicin domains to which it is
Domains in Colicins
437
attached. This evolutionary fine tuning may have been destroyed in some of our constructs. The hybrids that have the central domain (receptor binding site) of colicin El (AElA, AElEl) might bind to BtuB as colicin El does (that is, to a binding site different from that of colicin A) and perhaps this binding is not adapted to an OmpFdependent translocation monitored by the colicin A N-terminal domain. This hypothesis does not apply to the AAEl hybrid because the two domains involved in colicin A uptake are present. The explanation could be that the C-terminal domain of colicin A confers to the N-terminal and central domains a special conformation that makes the protein competent for translocation. At this stage, compatible combinations cannot be predicted. Results demonstrated that dependence upon OmpF, TolB and TolR was tightly related to the N-terminal domain of colicin A and dependence upon TolC was located in the N-terminal domain of colicin E 1. This dependence could be explained by an interaction between the N-terminal domain of colicin A and El with these To1 proteins. In addition, this demonstrates that the receptor-binding domain and the translocation domain did not function as a block but rather as individual entities. It has been reported that the translocation domain of colicin El comprised the polypeptide chain from residue 1 to 231 (Ohno-Iwashita & Imahori, 1982). Here, we demonstrate that ElAA, which has only the N-terminal 184 residues of colicin El, features the same characteristics as whole colicin El for translocation. Both BtuB and OmpF proteins have a binding site for colicin A (Cavard & Lazdunski, 1981; Fourel et al., 1990); however, while BtuB acts only as a receptor, OmpF is used both as a receptor and in translocation (Benedetti et al., 1989). In agreement with its role in translocation, we have demonstrated here that the binding for OmpF is not located in the central domain but in the amino-terminal domain of colicin A. This has been directly demonstrated by Bourdineaud et al. (1989). Under low ionic strength conditions, colicin A no longer requires BtuB for its reception, and bypass results clearly show that the N-terminal and central domains of colicin A are responsible for this property. Colicin A conformation seems to be different depending upon ionic strength (H. Benedetti, unpublished results). These two domains should, then, be sufficient to confer on colicin A its ionic strength-sensitive conformation. We next investigated whether the specific pore properties of whole colicin El and colicin A were conserved in the various hybrids. All hybrid proteins that had the C-terminal domain of colicin El (AAEl, AElEl and ElAEl) had the same channel characteristics as colicin El (no inactivation at alkaline pH at negative voltage, and fast pore closing at pH 5 at negative voltage). Hybrids containing only the C-terminal domain of colicin A (AElA and ElElA) featured channel
H. Benedetti
438
characteristics that were different from those of whole colicin A but were identical with those of the thermolytic fragment of colicin A. In contrast, the hybrid containing both the central and C-terminal domain of colicin A, i.e. El AA, featured the same characteristics as whole colicin A. This result shows that, the C-terminal domain of colicin A does not contain all the information needed to mimic the channel formed by the whole eolicin A. As previously reported, an interaction between the central and C-terminal domains of colicin A may be required to mimic the properties of the intact channel (Collarini et aZ., 1987; Prenette et al., 1989). The question of a specific recognition between the C-terminal domain of colicin A and colicin El and their immunity protein was also addressed in this study. The fact that ElElA and AAEl were inactive against cells expressing the immunity protein directed against colicin A and colicin El, respectively, confirmed this hypothesis. of hybrids the properties To conclude, constructed in this study have allowed us to demonstrate: (1) the binding site for OmpF is located in the N-terminal domain of colicin A; (2) OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) TolC dependence for colicin El is located in the N-terminal domain of colicin El; (4) the 183 N-terminal amino acid residues of colicin El are sufficient to promote ElAA uptake and thus probably colicin El uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that the domain interactions can be reconstituted in some hybrids (ElAA, ElElA and ElAEl), whereas in others (AAEl, AElEl and AElA) improper domain interactions may interfere with translocation; (7) there is specific recognition of the C-terminal domain of colicin
A and colicin
El
by their
own
immunity
protein. Structure-function relationships with the various hybrids will now be investigated using biophysical techniques (circular dichroism, etc.) to improve our understanding of their domain-domain interactions. We have also constructed hybrid proteins between colicins with different modes of action (colicin A, E2 and E3). The results of these studies will be published elsewhere. We thank M. Payan for careful preparation manuscript and the EMBO for providing H.B. short-term fellowship. This work was supported Centre National de la Recherche Scientifique Fondation pour la Recherche MBdicale. M.F. recipient, of a F.R.S.Q. fellowship.
of the with a by the and the was the
References Baty, D., Frenette, M., LloubBs, R., Geli, V. & Lazdunski, C. (1988). MoZ. MicrobioZ. 2, 807-811. Benedetti, H., Frenette, M., Baty, D., Lloub& R., Geli, V. & Lazdunski, C. (1989). J. Gen. Microbial. 135, 34133420.
et al. Bishop, L. J., Bjes, E. S., Davidson, V. L. Ss Cramer. W. A. (1985). J. Bacterial. 164, 237-244. Bourdineaud, J. P., Howard, 6. P. & Lazdunski, C. (1989). J. Bacterial. 171, 2458-2465. Cavard, D. & Lazdunski, C. (1979). Eur. J. Biochem. 96, 519-524. Cavard, D. & Lazdunski, C. (1981). PEMS Microbial. Letters, 12, 311-316. Coliarini, M., Amblard, G., Lazdunski, 6. & Pattus, F. (1987). Eur. Biophys. J. 14, 147-153. Cramer, W. A. & Phillips, S. K. (1970). J, Bacterial. 104, 819-825. Cramer, W. A., Daubert, J. R. & Uratani, Y. (1983). Bioehim. Biophys. Acta, 737, 173-193. Davidson, V. L., Brunden, K. R., Cramer, W. A. & Cohen, F. S. (1984). J. Membr. Biol. 79, 105-118. Davidson, V. L., Brunden, K. R., Cramer, W. A. $ Cohen, F. S. (1985). Proc. Nat. Acad. Sei., U.S.A. 82, 13861390. Davies, J. K. & Reeves, P. (1975). J. Bacterial. 123; 102-117. Fourel, D.; Hikita, C., Bolla, J. M., Mizushima, S. &, PagBs, J. M. (1990). J. Bacterial. 172; 36753680. Frenette, M., Knibiehler, M., Baty, D., Geli, V., Pattus, F.; Verger, R. & Lazdunski, C. (1989). Biochemistry, 28. 2509-2514. Frenette, M., Benedetti, H., Bernadac, A., Baty, D. & Lazdunski, 6. (1991). J. Mol. Biol. 217, 421428. Geii, V., Baty, Crozel, V., Morlon, J., Lloub$s, R., Pattus, F. 85 Lazdunski, C. (1986). X01. Gen. Genet. 202. 455-460.
Geli, V.; Baty, D.; Pattus, F. & Lazdunski, C. (1989). Ho2. MMicrobiol. 3, 679-687. Kagawa., Y. & Racker, E. (1971). J. Biol. Chem. 246, 5477-5479. Lazdunski, C. J., Baty, D., Geli, V., Cavard, D., Morlon? J.. Lloubits, R., Howard, S. P., Knibiehler, M., Chartier, M., Varenne, S., Frenette, M., Dasseux, 9. L. & Pattus; F. (1988). Biochim. Biophys. Acta, 947, 445-464. Lloub&s, R., Chartier, M., Journet, A., Varenne, S. & Lazdunski, C. (1984). Eur. J. Biochem. 114> 73-78. Mankovich, J. A., Lai, P. H., Gokul, N. & Konisky, J. (1984). J. Biol. Chem. 259, 8764-8768. Martinez, M. C., Lazdunski, C. & Pattus, F. (1983). EMBO J. 2, 1501-1507. Misra, R. & Reeves, P. R. (1987). J. Bacterial. 169, 4122-4130. Nagel de Zwaig, R. & Luria, S. E. (1967). J. Bacterial. 94, 1112-1123. Xomura., M. & Witten, C. (19673. J. Bacterial. 94: 10931111. Ohno-Iwashita, Y. & Imahori, K. (1982). J. Sol. Chem. 257, 6446-6451. C., Pattus, F., Cavard, D., Verger, R., Lazdunski: Rosenbusch, J. & Schindler, H. (1982). Tozicolz, 20, 205-206. Pattus, Ii‘., Cavard, D., Verger, R., Lazdunski: C., Rosenbusch, J. & Schindler, H. (1983a). In Physical Chenzistry of TTansmembrarte Ions Motions (Spaoh, G., ed.), pp. 407413, Elsevier Biomedical Preaa, Amsterdam. Pattus, F., Martinez, M. C., Dargent, B., Cavard, D., Verger, R. & Lazdunski, C. (1983b). Biochemistry f Wash.), 22: 5698-5703. Pugsley, A. P. (1989). In Protein Targeting, Academic Press, San Diego. Roos, U., Hartness, R. E. & Braun, V. (1989). Mol. Mierobiol. 3, 891-902.
Specijic
Properties
of Individual
Sabet, S. F. & Schnaitman, C. A. (1971). J. Bacterial. 108, 422-430. Schlinder, H. (1980). FEBS Letters, 122, 77-79. Schwartz, S. A. & Helinski, D. R. (1971). J. Biol. Chem. 246, 6318-6327.
Edited
Domains
in Colicins
439
Slatin, S. L. (1988). I&. J. Biochem. 20, 737-744. Sun, T. P. & Webster, R. E. (1987). J. Bucteriol. 109, 2667-2674. Tilby, M., Hindenmach, I. & Henning, U. (1978). J. Bacterial. 136, 1189-l 191.
by J. H. Miller
Biol.
(1991) 217, 429-439
Individual Domains of Colicins Confer Specificity in Colicin Uptake, in Pore-properties and in Immunity Requirement H. Benedetti’,
M. Frenette’Jf, D. Baty’, M. Knibiehler’ F. Pattus’ and C. Lazdunski’
‘Centre de Biochimie et de Biologic Mole’culaire de C.N.R.S. 31 Chemin Joseph Aiguier, B.P. 71 13402 Marseille Cedex 9, France 2European Molecular Biology Laboratory Postfach 10.2209, 6900 Heidelberg, Germany (Received 22 May
1990; accepted 17 September 1990)
Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and El. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin El is located in the N-terminal domain of colicin El; (4) the 183 N-terminal amino acid residues of colicin El are sufficient to promote ElAA uptake and thus probably colicin El uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin El by their respective immunity proteins.
1. Introduction The protein antibiotics colicin A and colicin El kill sensitive Escherichia coli by forming voltagedependent ion channels (Pattus et al., 1983a,b; Cramer et al., 1983; Davidson et al., 1984) thereby destroying the cell’s energy potential. They belong to the major group of colicins, which comprises colicins A, B, El, Ia, Ib, K and N, and are of particular interest to membrane biologists because they are unusual in being water-soluble transmembrane proteins. The vitamin B,, receptor, BtuB, is used by colicins A and El as a receptor protein at the surface of target cells, but colicin A also needs the OmpF porin for this step (Cavard & Lazdunski, 1981; Sabet & Schnaitmann, 1971). After binding to their receptors, colicins A and El translocate across the cell envelope to reach the cytoplasmic membrane and may interact with other membrane proteins during this step. Many mutations in the to1 locus (Nagel de Zwaig & Luria, 1967; Nomura & Witten, 1967; Davies & Reeves, 1975; Sun & Webster, 1987) render cells tolerant to t Present address: Dkpartement de Biochimie, Facultk des Sciences et de GEnie, Universitb Laval, Foy, Q&bee GlK 7P4, Canada.
Ste
these colicins and resistant to infection by filamentous bacteriophages (fl, Ml3 or fd), probably by blocking the transfer of colicins and DNA across the envelope. Colicin A requires TolA, TolB, TolQ and TolR with a low requirement for TolC (related to TolC effect on OmpF synthesis), while colicin El absolutely requires TolC, TolA and TolQ but neither TolB nor TolR (Nagel de Zwaig & Luria, 1967; Nomura & Witten, 1967). In addition, results reported by Benedetti et al. (1989) suggest that OmpF and BtuB play a part in colicin A and colicin El translocation, respectively. Thus, while both colicins use BtuB as a receptor, their uptake mechanism differs. It has been suggested that the amino-terminal domain of colicin A and of colicin El are involved in translocation (Ohno-Iwashita & Imahori, 1982; Martinez et al., 1983; Baty et al., 1988). One might therefore ask whether the specific features in the uptake of these two pore-forming colieins are related to their respective amino-terminal domains. The pore properties of colicin El and A have been extensively investigated using planar lipid bilayers and liposomes (Pattus et al., 1982, 1983a,b; Davidson et al., 1984, 1985). These properties differ with regard to extent significant to a
429 0022-2836/91/030429-11
%$03.00/O
0
1991 Academic
Press
Limited
H. Benedetti
430
pH-dependence of conductance and kinetics of closing after a voltage step. It would be of interest to investigate whether these properties are intrinsic properties of the C-terminal pore-forming domains. Finally, direct evidence for specific recognition between the C-terminal domain of colicin A and its corresponding immunity protein has never been provided. Colicin A immunity protein, designated Cai, is a 178 residue protein (Lloubes et al., 1984; Geli et al., 1986) with four transmembrane-spanning regions (Geli et al., 1989); it may interact with the pore-forming domain of cohcin A. To address these questions, six hybrid colicins containing various combinations of colicin A and colicin El domains were constructed (Frenette et al., 1990, accompanying paper). The hybrid colicins are designated by a three-letter code, in which the first letter corresponds to the N-terminal domain, the second to the central domain and the third to the C-terminal domain. For example, AElA means N-terminal domain of colicin A, central domain of colicin El and C-terminal domain of colicin A. The properties of these hybrids are presented here. The results demonstrate that dependence on TolB, TolR and OmpF for colicin A and TolC for colicin El is associated with their N-terminal domain. Furthermore, the immunity proteins only need the C-terminal domain of the corresponding colicin to specifically protect against their lethal action. Some hybrids turned out to have a very weak activity in viva while the activity in vitro was comparable to that of colicin A or El; thus, uptake of these hybrids appears to be affected and interactions between domains seem important for this step. As shown by assays in planar lipid bilayers, interactions between domains also affect the characteristics of the pore of colicin A. These interactions become even more critical when pore-forming colicins with different uptake systems (TonB and (H. Benedetti, Tol-dependent) are recombined unpublished result).
2. Materials and Methods (a) Bacterial strains and growth conditions The E. co& MC4100 strain was used as a host for the various colicinogenic plasmids to produce eolicins A, El and hybrid colicins for subsequent purifications. E. coli MC4100 strain (wild-type, OmpF- or BtuB(Cavard & Lazdunski, 1981)), E. co& K12 C600 strain (TolB- A593 (Sun & Webster, 1987) and TolC- ~602 (Davies & Reeves, 1975)) and E. coli GM1 strain (TPS 300 (orf 3 :: Cm) containing the toER mutation (Sun & Webster, 1987)) were used for the activity tests. They were grown in LB or M9 medium supplemented with thiamine (1 pg/pl) and 94 y0 (v/v) glycerol (for the fluorescence assay). MC4100 pColA9 and MC4100 pColE1 strains, immune to colicin A and El, respectively, were also used to test the activity of hybrid colicins. (b) Activity
ccssays
(i) Plate assay Dilutions from 10-l to 10-a of a 1 mg/ml stock solution of colicin A, coliein El and hybrid colicins were assayed
et al. by spotting lo-p1 portions on freshly seeded lawns of the MC4100 strain. The highest dilution for which a clear zone is observed on the MC4100 lawn is indicated in Table I. Activity of the hybrids has been assayed on lawns of the OmpF-, TolB-, TolC- and TolR- cells and cells carrying immunity to colicin A and El by spotting 10 ~1 of the stock solution.
(ii) Liquid culture assay To calculate the number of killing units, the SDS assay (Cavard & Lazdunski, 1979) was used on E. eoli strain MC4100. Cells of E. coli MC4100 were grown in LB medium at 37°C to A 6oo = 92. A volume of 100 yl of eolicin (A, El or hybrid) diluted in sodium phosphate buffer (10 mM-NaH,PO,/Na,HPO,, pH 6.8) containing @l% (v/v) Triton X-100 was added to 1.25 ml of this culture and incubated for 20 min at 37 “C with shaking, As a control, sodium phosphate was used instead of a colicin dilution. Subsequently, 100 ~1 of SDS (7.5 mg/ml) was added and turbidity at 600 nm was measured after 10 mm shaking at 37°C. The percentage of surviving cells was estimated from the turbidity ratio of the coliein-treated and the cont,rol samples. The number of killing units for each hybrid was calculated as described (Cavard &
Lazdunski, 1979). To test activity on OmpF- and BtuB- MC4100 ceils under normal conditions and to compare it to activity in bypass conditions, the routine assay was used. Cells of E. co& MC4100 were grown in LB medium to -4,,, = 02. A volume (10 id) of a colicin dilution (or sodium phosphate buffer as a control) was added to 100~1 of this culture and incubated for 20 min at 37°C with shaking. Subsequently, 1.25 ml of LB medium was added and the absorbance at 600 nm of the culture monitored until that of the control reached 0.4. The same number of killing units on MC4100 wild-type cells were obtained with this method a,nd the previous one. (iii) Assays in bypass conditions These tests were performed as described (Cavard $ Lazdunski, 1981). Conditions are the same as above except that the culture of E. coli MC4100 (wild-type. OmpF- or BtuB-) was grown to AeoO = 0.8, washed 3 times in IO mM-sodium phosphate buffer and diluted itat. this buffer to &,,e = 62. (iv) Fluorescence assays The bacteria were grown to 8 x IO* cells/ml in MM9 medium. The concentration of 8-anilinol-naphthalene sulphonic acid, magnesium salt (ANS) added to the cell suspension was %7x 10m5 w As described by Cramer & Phillips (1970), the excitation wavelength was 360 nm and the fluorescence emission was near 480 nm. After addition of AK’S, different amounts of colicins or hybrid colicins were added in a 1 ml cuvette (the volume added did not exceed 1.7% of the total) and fluorescence increase was measured as a function of time. The initial rate of fluorescence increase (which reflects membrane depolarization rate) was measured as a function of the amount of colicin or hybrid added in the cuvette. (c) Poye-forming
activity
in lipid planar
bilayers
Phosphatidylcholine from soybeans (Sigma Chemicai Co., St Louis, MO, type TIS) was purified according to Kagawa & Racker (1971). Bilayers were formed across a 150 pm hole pretreated with a 1 : 40 solution of hexadeeane in pentane from 1 mg/ml solutions of the phospholipid as described by Schlinder (1980).
Speci$c
Properties
of Individual
The membrane voltage was clamped to the desired value using 2 Ag/AgCl electrodes connected to the buffer in the 2 compartments via agar bridges. The membrane current was amplified using an I-V converter with an operational amplifier (Burr Brown 3528) and feedback resistors ranging from lo7 to lo9 R. The truns compartment was connected to the I-V converter and held at virtual ground potential. The sign of the membrane potential refers to the cis side of the membrane. Current was defined as positive when cations flowed into the tram compartment. Protein was always added to the cis side while the aqueous solution was stirred vigorously with a magnetic bar.
Domains in Colicins
431
Table 1 of hybrid colicins
Activity Colicins
Dilutionst
A
K.U./mg x lo-“1
lo4 lo4 5 2 2 lo4 lo3 lo4
El AElEl AElA AAEl ElAA ElAEl ElElA
8 15
N.D. N.D. N.D. 5 1.5 8
t Highest dilution (of a stock solution 1 mg/ml) for which a (d) Purification
of hybrid
colicins
The synthesis of colicins and hybrids was induced by adding mitomycin C to 300 rig/ml to growing cultures at A 6oo = 1.0. After 5 h of induction, the cultures were centrifuged at 10,OOOg for 15 min. For wild-type colicin A, mutated colicin A (encoded by pColA9 SX, see the accompanying paper) and AElA hybrid, supernatants of the centrifuged cultures were precipitated with 55% saturated ammonium sulphate overnight. The precipitate was centrifuged at 10,OOOg for 1 h. The pellet was resuspended and dialysed against 2 x 4 1 of 10 mw-phosphate buffer (Nap,), pH 68, 1 mM-EDTA. The dialysed material was applied on an S-Fastflow anionic exchanger (Pharmacia) in the same buffer, and proteins were eluted with a 0 M to @4 iv-Nacl gradient. Samples of the fractions were analysed by SDS/ polyacrylamide gel electrophoresis and immunoblotting, the fractions containing material reacting with anticolicin A monoclonal antibodies were pooled and dialysed against 25 mM-Mes (pH 55), 1 mM-EDTA. The dialysate was applied to a Mono S column (Pharmacia) and the proteins were eluted with a 0 M to @4 M-NaCl gradient. The fractions were analysed as desc@bed above. Fractions showing a &gle band on SDS/polyacrylamide gel electrophoresis Coomassie blue-stained gels that reacted with anti-colicin A monoclonal antibodies in immunoblotting were pooled, freeze-dried and stored at -20°C. For wild-type, mutated colicin El (encoded by pBRE1 XX, see the accompanying paper) and ElAEl hybrid, the centrifuged cells were extracted with 1 M-NaCl according to the method of Schwartz & Helinski (1971). The salt extract was dialysed against 2 x 4 1 of 10 mnrNaPi buffer (pH 7), 1 mM-EDTA and applied to a Mono Q column (Pharmacia) in the same buffer. The excluded fraction was dialysed against 50 miw-sodium borate (pH %5), 1 mm-EDTA; the dialysate was applied to a Mono S column (Pharmacia) and proteins were eluted with a 0 M to @4 M-NaCl gradient. Fractions showing a single band on SDS/polyacrylamide gel electrophoresis Coomassie blue-stained gels that reacted with anti-colicin El antibodies in immunoblotting were pooled, freezedried and stored at -20%. Hybrids ElAA, ElElA, AElEl and AAEl, which were not released from producing cells to the extracellular medium, were purified as follows. Centrifuged cells were sonicated in 10 mm-phosphate buffer (pH 6%; Nap,) containing 1 mM-EDTA (a metal protease inhibitor), 12 miw-N-dodecyl-fl, N-dimethyl-amino300 mM-NaCl, a-propane sulphonate (sulfobeta’ine SBl2), @02 mMphenylmethylsulphonyl fluoride (a serine protease inhibitor). The extract was centrifuged at 20,000 g for 20 min and the supernatant was dialysed against 2 x 4 1 of 10 mMNaP, buffer (pH 7) containing 1 mM-EDTA, 6 mi+-SBI2. The dialysate was applied to an S-Fastflow anionic
clear zone is observed on a lawn of MC1400 cells.
$ Comparison of the number of killing units (K.U.)/mg of colicins A, El and the most active hybrid colicins (tested as described in Materials and Methods). N.D. Not done.
exchange (Pharmacia) column in the Same buffer. Proteins were eluted with a 0 M to @4 M-NaCl gradient. Analysis of the fractions and the 2nd part of the purifications involving a chromatography on Mono S column were performed as described for colicin A and AElA but with the addition of 3 mM-SBl2 to each buffer.
3. Results (a) Activity
of hybrid
colicins
Purified colicins and hybrids were assayed on the sensitive indicator strain MC4100 for activity as described in Material and Methods. The results are presented in Table 1. The Ser/Val substitution at position 168 in colicin A and the Thr/Leu substitution at position 184 in colicin El, as well as the Arg/Lys substitutions at positions 394 and 379 in colicin A and colicin El, respectively, did not alter activity,
expression
or release
of mutated
colicins
(see the accompanying paper). Out of the six hybrids, all were active but to varying degrees. The activity of hybrids ElAA and ElElA was the same as that of the colicins A or El, where it was ten times lower with the hybrid ElAEl. But hybrids AElEl, AElA and AAEI were about lo4 times less active than colicins A and El (Table 1). The fluorescence assay enabled us to evaluate the rate of depolarization with hybrids and to compare these rates to those observed with colicins A and El. A typical example is shown in Figure 1. The initial rate of fluorescence increase (which reflects membrane depolarization rate) was measured as a function of colicin and hybrid concentrations and the dose-dependent rate was plotted (Fig. 2). Colicin A caused very fast membrane depolarization, while depolarization was much slower with colicin El. In addition, there was a marked difference between the two colicins with regard to the dose dependence of the rate. Using 1 pg of colicin A per cuvette, we obtained the same rate of depolarization as with 9 pg of colicin El per cuvette. For hybrids, behaviour in the kinetics of depolarization seemed to follow that of the C-terminal domain. Hybrids
H. Benedetti
432
eL al.
0.8 ? x 6 0.6 :: 6 2 0.4 .-5 z 0.2 z
0
5
IO
15
Colvins
20
25
(pg)
Figure 2. Variation of the initial rate of fluorescence increase as a function of the amount of colicin added; (o)A, (A) El, (m) ElAA. (0) ElElA, (0) ElAEl, (A) AElEl. The rate of fluorescence increase is expressed in relative
fluorescence/s.
r 0
1000
500 Time (5) (b)
Figure 1. Effect of colicin B or hybrid colicins on the fluorescence of APU‘S bound to E. coli MC4100 cells. Relative fluorescence at 480 nm is plotted as a function of time after the addition of colicin or hybrid colicins as described in Material and Methods. (a) Addition of colicin A: a, @05 pg; b, @I ,ug; c, 05 pg; d, 1 pg. (b) Addition of hybrid coiicins: a, 5pg of ElAEl; b, 0.5pg of ElAA; c, 5 pg of ElElA.
ElAA and ElElA caused fast depolarization at low concentration, like colicin A, while ElAEl caused slow depolarization even at high concentration. No information could be obtained from hybrids AElEl, AElA and AAEl because their low activity resulted in too weak an increase of fluorescence. The fact that every hybrid was active demonstrated that the translocation domain and the receptor-binding domain of colicins A and El could be recombined in a functional way, though with varying degrees of efficiency. We have observed that even though OmpF and BtuB act as receptor proteins for colicin A, they do not play exactly the same role; OmpF is involved both as a receptor and in translocation across the outer membrane, whereas BtuB is involved only at the receptor binding step (Cavard & Lazdunski, 1981; Benedetti et aZ., 1989). Tn contrast, for colicin El, OmpF is not involved either as a receptor or in translocation, while BtuB is involved in both processes. It was therefore of interest to assay the activity of hybrids on OmpF mutant cells in order to investigate which domain in colicin A conferred OmpF dependence. This was checked by plate and liquid cult’ure assays. The results are presented in Table 2. MC4100 OmpFstrain is still sensitive to colicin El at a dilution as high as 10-4 of a stock solution at a concentration of 1 mg/ml. As AElEl, AElA and AAEl hybrids are 10e4 times less active than colicin El, the 1 mg/ml stock solution of these hybrids should exhibit killing spots on OmpF- cells if this stra.in was sensitive. No killing spot was
observed, therefore the active hybrids AElEl, AElA and AAEl, which contained the N-terminal domain of coiicin A, were OmpF-dependent. This dependence was confined to the 168 N-terminal residues of colicin A since it was not observed with hybrids ElAA and ElAEl. TolB and TolR proteins are necessary for coliein A uptake but not for colicin El. In contrast, TolC protein is absolutely required for colicin El entry, whereas it is only partially needed for colicin A: probably because to02 caused a marked decrease in OmpF synthesis (Misra & Reeves 19873. Again, to investigate which domain was responsible for TolB, TolR, and TolC dependence, the activity of all hybrids was tested on TolB-, TolRand TolCstrains (Table 2). TolB- and TolR- cells were as sensitive to colicin El as wild-type MC4100 cells; thus; if they were sensitive to AElA, AAEl and BElEl hybrids, killing spots should have been observed. They were, on the other hand, fully sensitive to ElAA, EIEIA and ElAEl hybrids. These results clearly indicated
Activity of co&ins A and El and hybrid colicins ox ,rnutant cells (resistant or tolerant) and immune cells Activity
on difkrent
types of cells-i Cells immune to$
Colicins A El AElEl AElA AAEl EIAA ElElA ElAEl
OmpF + z +
ToIF + + + +
TolR-
ToiC-
Colicin A
Colicin El
t t f +
+ + + + -
+ + + +
i + + + -
i- + , Active; -. inactive. 1 The indicator strains were MC4100 pColAY and MC4100 pColE1 for assaying immunity to colicin A and El, respectively.
Specific
Properties
of Individual
100
100
in Colicins
433
-
80
ao-
60
60
40
40
20
20
0
Domains
0 I00
IO’
to*
103
IO4
100
105
10’
IO’
103
104
Id5
(6)
I00
2 a0 ;
80
60
60
z2 40
40
v) 20
20 0
100
IO’
IO”
I03
(b)
IO4
IO5
IO6
(e)
100
100
a0
a0
60
60
40
40
20
20
0 IO0
IO’
102
103
0 I04
105
100
IO’
I02
IO3
IO4
I05
Dilutions (cl
(f 1
Figure 3. Activity assays of hybrid colicins under normal and bypass conditions. The activity on MC4100 cells (wildtype, OmpF- and BtuB-) was tested in bypass and normal conditions as described in Materials and Methods. In each graph is indicated the colicin or hybrid tested in bold letters, and at the right the symbols for each curve. BC designates wild-type cells, BP bypass conditions, B- BtuB- cells and O- OmpF- cells. (a) AAEl activity assays under normal and bypass conditions. (b) Colicin A activity assays under normal and bypass conditions. (c) AElEl activity assays under normal and bypass conditions. (d) ElElA activity assays under normal and bypass conditions. (e) Colicin El activity assays under normal and bypass conditions. (f) ElAA activity assays under normal and bypass conditions. that TolB and TolR dependence was strictly related to the N-terminal domain of colicin A. TolC- cells were lo2 times less sensitive to colicin A than the MC4100 wild-type cells. Accordingly, the highest dilut)ion of colicin A stock solution (1 mg/ml) able to exhibit killing spots was 10m2. This suggested that 1 mg/ml solutions of AAEl , AEl A and AElEl (lo4 times less active than colicin A) should not produce any spot on a TolC- lawn even if they were active. In fact, they exhibited very turbid killing spots with a large diameter. The same turbid aspect was observed with dilutions of colicin A higher t,han 10’. As expected, colicin El stock solution (1 mg/ml) exhibited no spot and the sa’me results were observed with ElAA and ElElA (as active as colicin A or colicin El on wild-type cells) and ElAEl (10 times less active than colicin A or colicin El on wild-type cells). These results (Table 2) suggested that the N-terminal domain of colicin El is responsible for TolC dependence.
(b) Activity
of hybrid colicins shock conditions
under osmotic
It has been reported that resistant cells could be killed by colicins under osmotic shock conditions
(Cavard & Lazdunski, 1981; Tilby et al., 1978; Bishop et al., 1985; Benedetti et al., 1989). This technique allows BtuB receptor bypassing for colicin A, although limitation due to translocation (Tol-dependent step) across the envelope remains (Bourdineaud et al., 1989). In these conditions, the lethal action of colicin A is also enhanced. In contrast, the same conditions decrease the lethal activity of colicin El and BtuB cannot be bypassed. It was of interest to test the activity of the various hybrids in these conditions to determine whether one structural domain or more, and which were involved in these various features. ElElA and ElAEl behaved exactly like colicin El (Fig. 3(e) and (f)). There was a marked difference in sensitivity (lo-fold difference in dilution) between BtuB+ and BtuBcells under bypass conditions and both were OmpF independent because OmpFcells were as sensitive as wild-type cells under normal and bypass conditions. Furthermore, as observed with colicin El (Benedetti et al., 1989), the hybrids were less active on wild-type cells after the bypass treatment. As the only colicin El domain common to these two hybrids is the N-terminal domain, it is tempting to assign to this domain the features of colicin El in bypass conditions. However, the behaviour of the
434
H. Benedetti et al.
Table 3 Characteristics of colicins and hybrid colicins in bypass experiments Enhanced activity
No requirement for BtuB
Lower activity
Requirement for BtuB
Colicin A AAEl AElEl ElAA
Colicin A AAEl
Colicin El ElElA ElAEl
Colicin El AElEl AElA EIAA ElElA ElAEl
ElAA hybrid was not consistent with this hypothesis. As expected, the ElAA hybrid was OmpF independent. There was a marked difference in sensitivity (IO-fold difference in dilution) between BtuB+ and BtuB- cells in bypass conditions (Fig. 3(d)) but, like colicin A, it was more active on wild-type cells in bypass conditions than in normal conditions. Hybrids possessing the N-terminal domain of colicin A, AElEl, AElA and AAEl were lo4 times less active than colicin A in normal conditions. This is why (since in this assay colicin solution was diluted IO-fold by the cells) such a high survival percentage was observed for the wild-type cells in normal conditions. All these hybrids were more active on wild-type cells under bypass conditions than under normal conditions, like colicin A and ElAA hybrid. Furthermore, OmpF- cells were much less sensitive than wild-type cells under bypass conditions (Fig. 3(a) to (c)). Like colicin A, AAEl was as active on BtuBcells as on wild-type cells under bypass conditions (Fig. 3(a)). In contrast, BtuB seemed important for AElA and AElEl translocation, because cells deficient in this protein were less sensitive to the lethal action of the proteins than wild-type cells in bypass conditions. These results are summarized in Table 3. It clearly appears that the presence of both the
N-terminal and central domains of eolicin A is required for the bypass of BtuB protein under low ionic strength conditions. These two domains are then fully responsible for this colicin A charaeteristic. However, no precise domain could be held responsible for either a lower (like colicin El) OF higher-(like colicin A) activity on wild-type cells in bypass conditions compared to normal conditions. (c) Pore-forming ability of hybrids in lipid planar bilayers Extensive studies of channel properties of eolicin A and El have been carried out (see Slatin, 1988 and references therein). These studies demonstrated the existence of two different forms of channel with colicin A depending on pH. In contrast, only one form was observed with the C-t.erminal thermolytic fragment of colicin A. The ion channel properties of colicin El and its C-terminal fragment are more difficult to distinguish. In this study, we did not try to differentiate between colicin El and colicin El C-terminal domain type of channel properties. The hybrid colicins were thus classified into three categories according to their ion channel properties in lipid planar bilayers: eolicin A, colicin A C-terminal and colicin El classes. The major difference between the colicin El class and the two colicin A classes is the affinity for lipids at neutral pH. At pH 7.2, 94 ng of colicin A was sufficient to induce a large conductance increase when injected in front of lipid of planar bilayers (Fig. 4). In contrast, appearanee ion channel events occurred only when more than 1 pg of colicin El was injected under the sa,me conditions (Figs 6(b) and (c) and 7(a) to (e)). At neutral pH, a voltage step from 100 mV to 40 mV or negative potentials induces closing of colicin A channels (Fig. 5(b)). At the same ~33, the channels formed by the colicin A C-terminal peptide do not close at 40 mV and close slowly at negative potentials (Fig. 7(d)). The colicin El channels do not close at these potentials at neutral pH but close 70 mV
80 mv
90 mV 45 mV
(a) Figure 4. Pore-forming activity of the eoliein A type. Stepwise current increase after injection of @4 ng of (a) EPAA hybrid protein or (b) colicin A in front of an asolectin planar bilayer. The 2 proteins behave identically. The channels close after a voltage step to 45 mV or 70 mV (50 mw-Hepes (pH 7.2), 1 m-NH&L, 5 mmCaCl,). Broken lines indicate the zero-current level.
Xpeci$c Properties of Individual
Domains in Colicins
435
90 mV
PL_ 40 mV
(b)
(0)
Figure 5. Macroscopic conductances. The current relaxation kinetics after a voltage step from 90 mV to 40 mV are identical for (a) hybrid ElAA and (b) colicin A. Same conditions as in Fig. 4.
_---L. m
u _.____ _--_-_ _.__.. -._-_ m (cl 4_._.__ I min
Figure 6. Single channel events observed with different hybrids. (a) Hybrid AElA, 94 ng injected. This hybrid belongs to the colicin A C-terminal type of channel. The major single channel conductance is 40( +5) pS. (b) and (c) Hybrids ElAEl and AAEl, respectively; 2 pg injected. These hybrids belong to the colicin El class. The predominant single channel conductance is 30( k5) pS (50 mm-Hepes (pH 7.2) I M-NH&L, 5 mM-Call,). Broken lines indiea.te the zero-current level.
H. Benedetti
436
et al.
9q
90mVI
90~ -90
mV
-90
-90
mV
(a)
(b)
-90
mV
Cc)
mV
Cd)
Figure 7. Current traces in response to a voltage step. (a) and (c) AAEl, 10 pg injected. (b) AElEl, 10 pg injected. (d) AEIA, 10 ng injected. (c) and (d) 50 mM-Hepes (pH 7.2), 1 M-KH$L, 5 mM-Cd&. (a) and (b) 50 m&r-Tris-acetate (pH 5.0); 1 M-NH,CL, 5 mM-CaCl,. (a) and (c): note that AAEl conductance does not inactivate at pH 7.2 but inact’ivates quickly at pH 5.0 after a voltage step to - 90 mV. AAEl and AEl El belong to the colicin El type of channel. In contrast to colicin A and ElAA (Fig. 5), the hybrid AElA conductance inactivates slowly at -90 mV at neutral pH. This hybrid belongs to the colicin A C-terminal type of channel. Broken lines indicat,e the zero-current level. at negative potentials at acidic pH (Fig. 7(a) and (c)). Although the single channel conductances of colicin A and colicin El in NH,Cl at pH 7.2 differ significantly (40( f 5) pS and 25( 2 5) pS), they were not chosen to classify the hybrids due to the presence of sublevels of conductance, which render quantitative analysis rather difficult. Qualitative analysis of the hybrid protein conductances did not change the classification shown in Table 4. All the hybrid mutants, even those with low activity in wivo (AElEl, AElA and AAEI) were able to form ion channels in lipid planar bilayers with the same efficiency as the representatives of their categories. Among the hybrid mutants, ElAA is the only one that could not be distinguished from colicin A (Figs 4 and 5). The other mutants containing the C-terminal domain of colicin A (ElElA and AElA) formed channels similar to those formed by the colicin A C-terminal peptide (Figs 6(a) and 7(d)). These results strongly favour the hypothesis presented previously (Frenette et al., 1989) that the receptor-binding domain of colicin A may interact
with t,he pore-forming domain at neutral pHj affecting the channel gating by membrane potent’ial. As expected, the hybrids containing the poreforming doma,in of coliein El displayed low affinity for membranes at neutral pH and the same gating properties as colicin El (Table 4, Fig. 7(a) to (e)).
(d) The iwmunity in colicin A and colicin E:I :‘s spec$cally directed again& the C-terminal domabn It has been reported with the pore-forming coliein Ta and Ib that immunity is specifically directed against the C-terminal domain (Mankovich et al.. 1984). However, this had never been demonstrated with colicin A. Here, we observed that ElElA was inactive against cells producing immunity to eolicin A while, as expected, they were sensitive to AE7 El, AAEl and E1AEl. This result unambiguously demonstrates that immunity to colicin A (Cai protein) is directed against the C-t,erminaI domain of colicin A. A similar conclusion applied to colicin El as the hybrid AAEl was inactive against cells producing immunity to colicin El.
Table 4 BehaGour Sffinity membranes
Colicin
pH7
A El Th A$ AElEl SElA $AEl ElElA ElAA ElAEl
High Low High Low High Low High High Low
of hybrid
for
colicins
Inactivation
in planar at pH 7i
at +40
mv
++ ++ -
i -, So inactivation; + + , slow inactivation; + Th A, thermolysin fragment of colicin A.
-80
bilayers
lipid
Inactivation at pH 5t
IIll-
+++ ++ ++ ++ +++ + + + fast inactivation
- 90 mv
++ ++ ++ +++ ++ +++ ++ ++ +++
Type of pore Colicin Colicin Th A Colicin Th A Colicin Th A4 Colicin Colicin
A El El El B El
Xpecijk Properties of Individual
4. Discussion Colicins A and El share some homology in their C-terminal domain (Lazdunski et al., 1988). However, they differ to a large extent with regard to the sequence of the domains involved in receptor binding and translocation. Colicin El, indeed, requires TolC protein (an outer membrane protein) but not TolB, TolR nor OmpF for its action. In contrast, colicin A requires TolB, TolR, OmpF but not TolC (the low dependence probably reflects the effect of toZC on OmpF synthesis). It has been reported that colicin D (a protein synthesis inhibitor) that shares receptor specificity (Fep A) and TonB dependence for translocation with the pore-forming colicin B has an N-terminal and central domain very similar to that of this colicin. This result demonstrated that receptor and translocation domains for a pore-forming colicin are functional also for a colicin that has a cytoplasmic target (Roos et al., 1989). This suggested that the assembly of colicin genes occurred during evolution through recombination of DNA fragments encoding domains responsible for colicin activity and uptake. In this study along similar lines, we attempted to construct hybrid colicins derived from colicin A and colicin E 1. It was possible that by exchanging the N-terminal domains of colicin A and El, the requirements for TolB, TolR, TolC and OmpF could be exchanged. We also wanted to check whether the receptor-binding domain from a given colicin could be combined with the translocation domain of another colicin in a functional way, allowing penetration of the toxin into the cell. It might provide definite evidence as to whether there was specific recognition between the C-terminal domain of colicin A and colicin El and their immunity proteins, as with colicin Ia and Ib experimental et al., 1984). This (Mankovich approach assumed that domains were folded independently and that strong interactions were not required for a functional uptake. Six different constructs were made (Table 1). The six hybrids were found to be active on sensitive cells. Three of them, AEIEl, AElA and AAEl, exhibited very low activity in vivo. However, they were as active in vitro as the original colicins A or El constituting their C-terminal domain. This suggested that the uptake for these hybrids was a limiting step. It is striking that these three hybrids have the N-terminal domain of colicin A and use OmpF for translocation. These features may be compa.red to those of secretory proteins. In this case, it has been demonstrated that their conformation is a constraint on the routing and translocation of proteins. Extensive studies on different combinations of signal peptides and secretory proteins have strongly suggested that not all combinations are compatible (Pugsley, 1989). Therefore, with colicins there is a possibility that each amino-terminal domain has specifically evolved to be most compatible with the two other colicin domains to which it is
Domains in Colicins
437
attached. This evolutionary fine tuning may have been destroyed in some of our constructs. The hybrids that have the central domain (receptor binding site) of colicin El (AElA, AElEl) might bind to BtuB as colicin El does (that is, to a binding site different from that of colicin A) and perhaps this binding is not adapted to an OmpFdependent translocation monitored by the colicin A N-terminal domain. This hypothesis does not apply to the AAEl hybrid because the two domains involved in colicin A uptake are present. The explanation could be that the C-terminal domain of colicin A confers to the N-terminal and central domains a special conformation that makes the protein competent for translocation. At this stage, compatible combinations cannot be predicted. Results demonstrated that dependence upon OmpF, TolB and TolR was tightly related to the N-terminal domain of colicin A and dependence upon TolC was located in the N-terminal domain of colicin E 1. This dependence could be explained by an interaction between the N-terminal domain of colicin A and El with these To1 proteins. In addition, this demonstrates that the receptor-binding domain and the translocation domain did not function as a block but rather as individual entities. It has been reported that the translocation domain of colicin El comprised the polypeptide chain from residue 1 to 231 (Ohno-Iwashita & Imahori, 1982). Here, we demonstrate that ElAA, which has only the N-terminal 184 residues of colicin El, features the same characteristics as whole colicin El for translocation. Both BtuB and OmpF proteins have a binding site for colicin A (Cavard & Lazdunski, 1981; Fourel et al., 1990); however, while BtuB acts only as a receptor, OmpF is used both as a receptor and in translocation (Benedetti et al., 1989). In agreement with its role in translocation, we have demonstrated here that the binding for OmpF is not located in the central domain but in the amino-terminal domain of colicin A. This has been directly demonstrated by Bourdineaud et al. (1989). Under low ionic strength conditions, colicin A no longer requires BtuB for its reception, and bypass results clearly show that the N-terminal and central domains of colicin A are responsible for this property. Colicin A conformation seems to be different depending upon ionic strength (H. Benedetti, unpublished results). These two domains should, then, be sufficient to confer on colicin A its ionic strength-sensitive conformation. We next investigated whether the specific pore properties of whole colicin El and colicin A were conserved in the various hybrids. All hybrid proteins that had the C-terminal domain of colicin El (AAEl, AElEl and ElAEl) had the same channel characteristics as colicin El (no inactivation at alkaline pH at negative voltage, and fast pore closing at pH 5 at negative voltage). Hybrids containing only the C-terminal domain of colicin A (AElA and ElElA) featured channel
H. Benedetti
438
characteristics that were different from those of whole colicin A but were identical with those of the thermolytic fragment of colicin A. In contrast, the hybrid containing both the central and C-terminal domain of colicin A, i.e. El AA, featured the same characteristics as whole colicin A. This result shows that, the C-terminal domain of colicin A does not contain all the information needed to mimic the channel formed by the whole eolicin A. As previously reported, an interaction between the central and C-terminal domains of colicin A may be required to mimic the properties of the intact channel (Collarini et aZ., 1987; Prenette et al., 1989). The question of a specific recognition between the C-terminal domain of colicin A and colicin El and their immunity protein was also addressed in this study. The fact that ElElA and AAEl were inactive against cells expressing the immunity protein directed against colicin A and colicin El, respectively, confirmed this hypothesis. of hybrids the properties To conclude, constructed in this study have allowed us to demonstrate: (1) the binding site for OmpF is located in the N-terminal domain of colicin A; (2) OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) TolC dependence for colicin El is located in the N-terminal domain of colicin El; (4) the 183 N-terminal amino acid residues of colicin El are sufficient to promote ElAA uptake and thus probably colicin El uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that the domain interactions can be reconstituted in some hybrids (ElAA, ElElA and ElAEl), whereas in others (AAEl, AElEl and AElA) improper domain interactions may interfere with translocation; (7) there is specific recognition of the C-terminal domain of colicin
A and colicin
El
by their
own
immunity
protein. Structure-function relationships with the various hybrids will now be investigated using biophysical techniques (circular dichroism, etc.) to improve our understanding of their domain-domain interactions. We have also constructed hybrid proteins between colicins with different modes of action (colicin A, E2 and E3). The results of these studies will be published elsewhere. We thank M. Payan for careful preparation manuscript and the EMBO for providing H.B. short-term fellowship. This work was supported Centre National de la Recherche Scientifique Fondation pour la Recherche MBdicale. M.F. recipient, of a F.R.S.Q. fellowship.
of the with a by the and the was the
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Specijic
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Edited
Domains
in Colicins
439
Slatin, S. L. (1988). I&. J. Biochem. 20, 737-744. Sun, T. P. & Webster, R. E. (1987). J. Bucteriol. 109, 2667-2674. Tilby, M., Hindenmach, I. & Henning, U. (1978). J. Bacterial. 136, 1189-l 191.
by J. H. Miller