Proc. NatI. Acad. Sci. USA Vol. 85, pp. 7521-7525, October 1988 Biochemistry Subunit S1 of pertussis : Mapping of the regions essential for ADP-ribosyltransferase activity (erts toxin/ADP-rlbosylatin/subunlt Sl/site-directed mutagenesis) MARIAGRAZIA PIZZA, ANTONELLA BARTOLONI, ANNA PRUGNOLA, SERGIO SILVESTRI, AND RINo RAPPUOLI Sclavo Research Center, Via Fiorentina 1, 53100 Siena, Italy Communicated by A. M. Pappenheimer, July 11, 1988 (receivedfor review April 8, 1988)

ABSTRACT The toxicity of is mediated by related, nontoxic molecules that can be used as vaccines (20- the ADP-ribosyltransferase activity of subunit S1. To under- 22). stand the structure-function relationship of'subunit Si and For and Pseudomonas , a series of stud- guide the construction of nontoxic molecules suitable for ies, which include nitrosoguanidine mutagenesis ofthe vaccines, we constructed and expressed in Escherichia coli a (23), characterization of the mutant genes (5) and their series of amino-terminal and carboxyl-terminal deletion mu- products (24-26), photoaffinity labeling of the toxins with tants as well as a number of molecules containing NAD (27, 28), site-directed mutagenesis (29, 30), and crys- substitutions. The shortest still retaining enzymatic tallographic structure of the Pseudomonas A (31) activity contains amino-acids 2-179. Within this region we have led to the identification of amino acids essential to the identified three mutants in which amino acid substitutions enzymatic activity of the . For example, Glu-148 of abolish the enzymatic activity. Mutation of amino acids 8 and and Glu-553 of Pseudomonas -exotoxin A, 9 or.50 and 53, located within the region of the S1 subunit of located within the catalytic site of the two , cannot pertussis toxin homologous to , causes loss of be replaced even with an aspartic acid without abolishing enzymatic activity. Outside this homology region, substitution enzymatic activity (29, 30). of Glu-129 with glycine or aspartic acid also eliminates the We used a similar approach to study the structure-function enzymatic activit of the Si subunit. In this respect, Glu-129 relationship ofthe S1 subunit ofpertussis toxin. By carboxyl- resembles the glutamic acid that is crucial for the catalytic and amino-terminal deletion analysis and site-directed mu- activity of diphtheria and Pseudomonas toxins. Once intro- tagenesis, we identified at least three regions of the S1 duced into the Bordetela perussis , the above subunit that are essential for function. Substitutions mutations should lead to the synthesis of nontoxic pertussis within these regions produce enzymatically inactive mole- toxin molecules suitable for vaccine production. cules. ADP-ribosylation of the target substrates in eukaryotic cells MATERIALS AND METHODS is a common mechanism of action of many bacterial toxins (1-3). The best-studied molecules that adopt this Construction of the Si Deletion Mutants. The S1 subunit of mechanism are diphtheria toxin (4-7), Pseudomonas exotox- pertussis toxin was expressed in Escherichia coli fused to the in A (8-10), cholera toxin (11-13), and pertussis toxin (14- 98 amino-terminal amino acids of the MS2 polymerase. This 16), but several other toxins possessing ADP-ribosyltrans- fusion protein (PTE255) contains amino acids 2-235 ofthe S1 ferase activity have also been described (17, 18). The toxins subunit and is enzymatically active (32). The coding for usually contain two functional moieties: A, which is enzy- the amino acids 2-235 is contained within a BamHI-Xba I matically active, and B-, which recognizes and binds the fragment flanked by an EcoRI site at the 5' end and a HindIII receptors on the surface facilitating the entry of the site at the 3' end. To obtain the plasmids expressing carboxyl- enzymatically active subunit into the target cells. terminal deletion mutants of the S1 protein, the plasmid The proteins that are ADP-ribosylated by diphtheria, pTE255 was digested first with Xba I and then with Nco I, Pseudomonas, cholera, and pertussis toxins are GTP-binding Nru I, BalI, Sal I, and Sph I, respectively.' The sticky ends proteins involved in protein synthesis (diphtheria and Pseu- generated by the restriction enzymes were then repaired by domonas toxins) or in the transfer of signals through the the large fragment of DNA polymerase, and the plasmids membrane of eukaryotic cells (cholera and pertussis toxins) were circularized by DNA ligase. During this process, the (1, 2). In spite of the common mechanism of action of these natural stop codon of the S1 subunit was lost, and therefore toxins, little or no similarity has been detected by computer the new proteins contained a few amino acids fused at the analysis of their primary structures: only a short amino- carboxyl terminus: NCO, NRU, and SPH proteins had the terminal similarity could be found between fragment A of following carboxyl-terminal unrelated amino acids: Leu-Pro- cholera toxin and the S1 subunit of pertussis toxin (15, 16). Arg-Ala-Phe-Arg. BAL and SAL proteins contained, respec- However, after mapping the functional domains ofdiphtheria tively, 50 and 16 amino acids deriving from the sequence of and A, a significant similarity has pBR322 (33) fused at the carboxyl terminus. To generate the been discovered between the functionally equivalent regions amino terminal deletion mutants, the plasmid pTE255 was cut in the two enzymes (19). with BamHI and then with Sph I, Sal I, and Bal I, respec- A better understanding of the relationship between the tively. The sticky ends were then repaired by the large structure and function of these proteins should help clarify fragment of DNA polymerase, and the fragment was ligated the mechanism of action of the toxins and provide a sound by DNA ligase. By this manipulation, the deleted S1 gene was theoretical basis for the construction of immunologically inserted in the same frame of the MS2 polymerase. Mutants 34A and NCO/BAL were obtained by a similar procedure, the with BamHI/BstNl and The publication costs ofthis article were defrayed in part by page charge cutting plasmid pNCO payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: mAb, monoclonal antibody. 7521 Downloaded by guest on October 2, 2021 7522 Biochemistry: Pizza et al. Proc. Natl. Acad. Sci. USA 85 (1988) BamHI/BalI, respectively. The amino acids of the S1 sub- glutamic acid; 31, TACTCCGTTTTCGTGGTC, which unit, which are expressed by each ofthe deletion mutants, are changed Thr-159 to ; 31G, GAATAGGCCGTGGTCG- shown in Fig. 1. The M, values for the fusion proteins are as TG, which changed Glu-160 to glycine; 28D, GCCAGA- follows: NCO, 35,000; NRU, 31,500; BAL, 31,100; SAL, TAGTCGCTCTGG, which changed Glu-129 to aspartic acid; 24,300; SPH, 19,200; 16A, 35,500; 34A, 29,300; SPH/HIND, 1617, the mutations 16 and 17 were combined, and, therefore, 29,200; 255/SAL, 24,700; 255/BAL, 23,100; NCO/BAL, in this mutant Asp-109 and Ala-124 are changed to glycine 20,400; and 18, 20,000. and aspartic acid, respectively. The presence of the unrelated amino acids did not have ADP-Ribosylation. The mutant proteins were assayed for detectable effects on the enzymatic activity (in the case of their ability to ADP-ribosylate transducin as already de- mutants NCO and NRU) or on the stability of the recombi- scribed (32, 36). In particular, after temperature induction of nant molecules; in fact, all ofthem gave only one major band a 10-ml culture, the inclusion bodies were prepared (32), and in sodium dodecyl sulfate/polyacrylamide gel stained with the partially purified fusion proteins were resuspended in 200 Coomassie blue, which, with the exception ofmutant 18, was ,ul of 8 M urea. Five microliters were then used for the recognized by one of the monoclonal antibodies (niAbs) ADP-ribosylation reaction; after activation in a mixture (Table 1). The immunological reactivity of mutant 18 was containing 5 .d ofurea extract, 15 ,ul of water, and 7 /1 of 0.1 confirmed by immunologic blotting by use of a rabbit poly- M dithiothreitol for 30 min, 5 pl of 2 M Tris, pH 7.5, 1 td of clonal antiserum against a synthetic peptide covering the 100 mM ATP, 1 t4 of 10 mM GTP, 10 !d of0.25 M thymidine, region 172-194. The folding of the recombinant molecules 10 ,ul of retinal outer segment membranes (ROS), and 80 p1 also seemed to be unaffected because after in vitro refolding, ofH20 were added. The reaction mixture was then incubated the truncated fragments could generate a conformational at room temperature for 2 hr and centrifuged; the insoluble epitope that requires the interaction of the amino- and pellet containing the transducin was solubilized in a sodium carboxyl-terminal parts of the molecule (34). dodecyl sulfate-loading buffer and was loaded in a 12.5% Site-Directed Mutagenesis. To mutagenize the S1 gene, the acrylamide gel. After electrophoresis, the labeled transducin restriction fragment BamHI-Xba I ofplasmid pTE255, which was visualized by autoradiography. For quantitative analy- contains the S1 gene, was subcloned in Bluescript-KS (Stra- sis, the bands containing transducin were cut out, and tagene, San Diego, CA), and the single-stranded DNA was radioactivity was counted in a 13 counter. mutagenized using oligonucleotide primers as described (35). NAD+ Glycohydrolase Activity. The samples in 8 M urea After mutagenesis, the BamHI-Xba I fragment was sub- were prepared as for the ADP-ribosylation reaction. Then, 5 cloned again into the expression vector pEX34b (32) for the 1d were diluted in the reaction mixture and incubated for 18 expression of the mutant protein. The following oligonucle- hr at 30°C. The composition of the reaction mixture was 10 otide primers were used: 41, GTCATAGCCGTCTACGGT, mM dithiothreitol/100 mM Tris, pH 7.5/1 mM ATP/0.1 mM which changed Tyr-8 and Arg-9 to aspartic acid and glycine, GTP/25 mM thymidine/1 jA of [carbonyl-14C]NAD respectively; 22, TGGAGACGTCAGCGCTGT, which (=z100,000 cpm) in afinal volume of 100 p1. The nicotinamide changed Phe-50 to glutamic acid [during this mutagenesis an was then separated from the nonhydrolyzed NAD by thin- additional mutation (Thr-53 -. Ile) was added by the DNA layer chromatography; 2 ,ul of the reaction mixture were polymerase]; 25, CTGGCGGCTTCGTAGAAA, which loaded on nitrocellulose thin-layer plates, which were devel- changed Gly-99 to glutamic acid; 17, CTGGTAGGTGTC- oped with isobutyric acid/ammonium hydroxide/water, CAGCGCGCC, which changed Asp-109 to glycine; 26, 63:1:33 (37). Qualitative analysis was done by autoradiogra- CGCCACCAGTGTCGACGTATTCGA, which changed phy ofthe thin-layer plates; for quantitative analysis the plate Tyr-111 to glycine. During the mutagenesis, part of the was divided into 1-cm segments, the cellulose was scraped off oligonucleotide sequence was duplicated so that this latter the thin-layer plate, and radioactivity was counted in a j3 mutant, in addition to the Tyr 111 -+ Gly change, had four counter. additional amino acids (Asp-Thr-Gly-Gly) inserted in posi- Immunoassays. The production and the stability of the tion 113. Further mutations and primers included the follow- recombinant molecules was monitored by Coomassie blue ing: 27, GCCAGCGCTTCGGCGAGG, which changed Gly- staining of sodium dodecyl sulfate/polyacrylamide gels and 121 to glutamic acid; 16, GCCATAAGTGCCGACGTATTC, confirmed by immunologic blotting (38). For this purpose, we which changed Ala-124 to aspartic acid; 28, GCCAGA- used two nonprotective mAbs, X2XS and 6G7, which rec- TACCCGCTCTGG, which changed Glu-129 to glycine; 29, ognize linear epitopes located within the first 60 amino acids GCGGAATGTCCCGGTGTG, which changed Arg-135 to and in proximity to amino acid 143, respectively (34, 39). The

0 - m 50 100 11 " ,00 z 200 Z ------I---0------I------0------f ---AI ----- 51 I 1 Opp E r PL t r p U 1 7 St p T P 62T0N0S 06 E I E 6C ImDTo E 0 6 E 41 22 25 17 26 27 16 28 29 30 306 0 260

NCO

------SL SAL WPH

164 ------346 0061N0 255/SAL 26/A3 N0O0/S 16 FIG. 1. Amino acid sequence of the subunit S1 of pertussis toxin (S1 BP) and summary of the mutants used in this study. Below the amino acid sequence we report the amino acids that differ in the S1 subunit of parapertussis (S1 BPP) and Bordetella bronchiseptica (S1 BB). The amino acids that were changed by site-directed mutagenesis are reported in the mutants line. Each mutant is identified by the number reported below the amino acid changed. The dotted lines represent parts of the S1 molecule that are expressed by each carboxyl-terminal or amino-terminal deletion mutant. Above the amino acid sequence are listed the restriction enzymes, which cut the S1 gene only once and were used to construct the deletion mutant genes. Downloaded by guest on October 2, 2021 Biochemistry: Pizza et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7523 Table 1. Summary of the properties of the recombinant fragments of the S1 subunit ADP- NAD- Binding of Binding of non- Recombinant ribosylation glycohydrolase* protective protective mAbs fragment activity, % activity, % mAbst 6G7 X2XS S1BP (pTE225) 100 100 + + + S1BPP 100 + + - + S1BB 100 + + + + NCO 100 48 + + + NRU 70 26 + + + BAL / <5 - - + SAL / <5 - _ + SPH / <5 - _ + 16A / <5 - + - 34A / <5 - + - SPH/HIND / <5 - + - 255/SAL / <5 - + - 255/BAL / <5 - + - NCO/BAL / <5 - + - 18 / <5 - - - 25 100 + + + + 31 100 + + + + 29 92 48 + + + 26 150 + + + + 27 42 + + + + 17 46 + + + + 16 50 + + + + 1617 23 + + + + 31G 70 + + + + 41 / <5 - + + 22 / <5 + + + 28 /<5 + + + 28D / <5 + + + * +, Glycohydrolytic activity was present, but quantitative analysis was not performed; <5, Quantitative analysis was performed, and the activity was undetectable (<5% is background level). tEffect of three protective mAbs: 1B7, 3F10, and 6D2X11C, which gave identical results. ability of the recombinant molecules to assume the native to ADP-ribosylate transducin, for NAD+ glycohydrolase conformation ofthe S1 subunit was analyzed in immunologic activity (34, 35), and for immunoreactivity with the mAbs blots by using three protective mAbs as a probe: 1B7, 3F10, (Table 1). and 6D2X11C, obtained from H. Sato (40, 41) and D. Burns As shown in Fig. 2A and Table 1, the protein NCO, which (Food and Drug Administration, Bethesda, MD), respec- tively. We have, in fact, shown that these protective mAbs . W. Z bind only to a conformational epitope that requires the U-1 7 interaction of the amino terminal and the carboxyl terminal ;L; -1-1 < Z. 'IC Ir Z. Ir. tr,r - parts of the S1 molecule and that the ability of recombinant z z tr C.,. ?I,, cr. IA ,.'zt-. fragments to assume the native conformation can be tested directly in an immunologic blot using one ofthese mAbs (34). In this paper, protective or nonprotective indicates only the ability of these antibodies to protect the Chinese hamster ovary cells from pertussis toxin activity. However, one ofthe antibodies used can also protect mice from the intracerebral challenge with virulent B. pertussis (40, 41). If) 0 Utr RESULTS w Z IC, ;- C, %C ;; f--t t-F-. Y0 as- -- e4 QCoc cx Construction of Carboxyl-Terminal and Amino-Terminal r- e4 o1 ell - Deletion Mutants. We showed that the S1 subunit ofpertussis toxin can be efficiently expressed in E. coli fused to the 98 amino-terminal amino acids of the polymerase of phage MS2 (32). The resulting protein (PTE255) containing amino acids 2-235 of the S1 subunit shows an enzymatic activity compa- rable to that of pertussis toxin. To map the regions of the S1 molecule that are essential for enzymatic activity, the plas- mid pTE255 was cut with suitable restriction enzymes (Fig. FIG. 2. Autoradiography of a sodium dodecyl sulfate/polyacryl- 1) or treated with exonuclease Bal-31, to obtain a series of amide gel for the assay of the ADP-ribosylation of transducin with plasmids expressing only part ofthe S1 molecule. The region [32P]NAD. (A) Carboxyl-terminal and amino-terminal deletion mu- of the S1 molecule expressed by each mutant clone is shown tants. (B) Mutants obtained by site-directed mutagenesis of the S1 in Fig. 1. The proteins obtained were tested for their ability gene. Downloaded by guest on October 2, 2021 7524 Biochemistry: Pizza et al. Proc. Natl. Acad. Sci. USA 85 (1988) lacks amino acids 211-235, still retains full ADP-ribosyltrans- mutagenesis were all recognized by the protective mAbs ferase activity and about 50% of the NAD glycohydrolase except for mutant 41 (Table 1). activity. Protein NRU, which lacks amino acids 180-235 also retains =70% ofthe ADP-ribosyltransferase activity and 25% DISCUSSION of the NAD+ glycohydrolase activity. On the other hand, amino-terminal deletion of only 10 amino acids (protein 16A) Pertussis toxin is a of B. pertussis and the abolishes both enzymatic activities. None of the other car- main component of an acellular vaccine against whooping boxyl-terminal or amino-terminal deletion mutants show any cough (48, 49). Complete detoxification of pertussis toxin is enzymatic activity. We conclude that the carboxyl terminal necessary for vaccine production, as even trace amounts of amino acids 180-235 are not necessary for the two enzymatic active pertussis toxin can produce the severe side effects activities, whereas the amino-terminal amino acids are es- observed at a frequency of 10-5 in the vaccinated popu- sential. lation (50, 51). The pioneering work with diphtheria cross- Site-Directed Mutagenesis of the Si Subunit. To identify reacting materials showed that the best way to produce a amino acids essential to the enzymatic activity, we have used completely detoxified molecule was to engineer its gene so site-directed mutagenesis. The choice of the amino acids to that that gene encodes an immunologically active but non- be mutagenized was based on previous studies carried out toxic molecule (52, 53). Failure to induce any protective with diphtheria and Pseudomonas toxins, in which modifi- against B. pertussis by immunizing with the recom- cation or substitution of charged, aromatic, or small nonpolar binant pertussis toxin subunits produced in E. coli (32) amino acids has been shown to impair or abolish the enzy- suggests that here also an enzymatically inactive holotoxin matic activity (29, 30, 42-46). Furthermore, the comparison should be used in constructing new vaccines against whoop- of the sequence of the S1 subunit of pertussis toxin with that ing cough. The aim ofthis study was to identify enzymatically of the homologous proteins of Bordetella parapertussis and inactive S1 molecules the genes for which can be introduced Bordetella bronchiseptica (Fig. 1) shows that several amino into the B. pertussis chromosome to direct synthesis of acids can be substituted without any impairment of the nontoxic pertussis toxin molecules. enzymatic activity (47). Because most of these substitutions We identified two mutants (22 and 28) that should be occur after amino acid 161, we felt justified in confining the suitable to produce a safe vaccine against . mutagenesis upstream from this region. Amino acids located Both mutants are recognized by mAbs capable ofneutralizing within the regions homologous to cholera toxin were prefer- the native toxin, but these mutants have lost the enzymatic entially mutagenized. In general, to introduce drastic activity ofthe S1 subunit. Mutant 41, enzymatically inactive, changes, polar-charged amino acids were replaced by small seems to have also lost the correct folding necessary to create nonpolar amino acids and vice-versa, whereas aromatic the epitope that binds and generates neutralizing antibodies. amino acids were either changed to glycine or to charged Our data confirm and extend the findings of Locht et al. amino acids. After site-directed mutagenesis of the S1 gene, (54) and Bums et al. (55) showing that removal of the region we obtained 13 mutant proteins (Fig. 1), which were ex- from amino acids 187-235 ofthe S1 subunit does not affect the pressed in E. coli as fusion proteins. Alteration of the ADP-ribosylating activity (54) and that proteolytic fragments enzymatic activity was seen in most of the mutants (Table 1 of the S1 subunit, lacking the carboxyl-terminal region, still and Fig. 2). Only two mutants (25 and 31) retained full retain the glycohydrolytic activity (55). We restricted the enzymatic activity. Mutant 26 showed increased enzymatic enzymatically active portion of the S1 subunit to a region activity, whereas in all others the activity was reduced. spanning from amino acid 2 to 179. Mutants 41, 22, and 28 lost all enzymatic activity. Inactiva- Within this fragment we identified three mutants in which tion of the S1 subunit after the substitution of Glu-129 by amino acid substitutions abolish the enzymatic activity. Two glycine (mutant 28) recalls a similar observation made with of them (mutants 41 and 22) are located within the regions diphtheria and Pseudomonas toxins, in which the glutamic homologous to cholera toxin (15, 16), confirming that these acid located within the catalytic site could not be changed conserved sequences are important in the enzymatic activity. without complete loss of the enzymatic activity (29, 30). The third one (mutant 28) suggests that Glu-129 corresponds Further indications that Glu-129 is equivalent to Glu-148 of to Glu-148 of diphtheria toxin and Glu-553 of Pseudomonas diphtheria toxin and to Glu-553 of Pseudomonas exotoxin A exotoxin A. Note that on comparison of the primary se- is given by mutant 28D, in which Glu-129 is replaced by quences, two other glutamic acids seemed more likely to be aspartic acid. The mutant protein expressed in E. coli shows crucial for the catalytic activity: Glu-160, the surrounding no ADP-ribosyltransferase or glycohydrolytic activity (Table sequence Glu-Thr-Thr-Thr-Thr-Glu-Tyr-Ser-Asn of which is 1 and Fig. 2). homologous to the region containing Glu-148 of diphtheria Interaction of the Mutant Proteins with mAbs. Alteration of toxin (Glu-Gly-Ser-Ser-Ser-Val-Glu-Tyr-Ile-Asn) and Glu- the enzymatic activities of the mutant proteins described can 210, which, as in the case of diphtheria and Pseudomonas be due to the substitution of amino acids involved in the exotoxin A, is followed by a tryptophan after four amino enzymatic activity, to conformational changes of the mole- acids. cule, or to both. The conservation of the native structure of We show here that mutagenesis of Glu-160 or deletion of the S1 subunit in the enzymatically inactive mutants is the region containing Glu-210 does not abolish the enzymatic important because our final aim is the construction of activity. Among the other mutants, it is notable that although nontoxic pertussis toxin molecules for vaccination. We have the mutagenesis of Tyr-lil and an insertion of four amino recently shown that the S1 subunit contains only one major acids in position 113 increases the enzymatic activity by 50%o protective epitope, which requires the interaction of the (mutant 26), Black et al. (56) recently showed that a similar amino-terminal and the carboxyl-terminal part of the mole- insertion of four amino acids in position 107 reduces the cule and that the ability of the mutants to assume the native enzymatic activity to 1%, indicating that the sequences conformation can be tested in an immunologic blot with these immediately upstream of amino acid 110, but not those protective antibodies (34). downstream from it, are crucial in the catalytic activity. Table 1 shows that these mAbs recognize only the enzy- Further evidence for the critical role ofthe regions surround- matically active deletion mutants, indicating that as for the ing Tyr-107 and Glu-129 is given by mutants 17, 27, and 16, enzymatic activity, formation of the protective epitope re- which have only 50% of their original activity. The two quires amino acids 2-10 and those contained between sites regions seem independent, as the combination of mutations BalI and Nru I. The mutants obtained by site-directed 16 and 17 further decreases the activity to 25% (mutant 1617). Downloaded by guest on October 2, 2021 Biochemistry: Pizza et aL Proc. Natl. Acad. Sci. USA 85 (1988) 7525 In addition to the enzymatically active domain, the S1 24. Uchida, T., Pappenheimer, A. M., Jr., & Greany, R. (1973) J. subunit has a binding site for the subunits S2-S5. The region Biol. Chem. 248, 3838-3844. of amino acids 180-235, which is not required for enzymatic 25. Uchida, T., Pappenheimer, A. M., Jr., & Harper, A. A. (1973) J. Biol. Chem. 248, 3845-3850. activity, probably mediates this interaction. In agreement 26. Uchida, T., Pappenheimer, A. M., Jr., & Harper, A. A. (1973) with this hypothesis, Bums et al. (55) have shown that J. Biol. Chem. 248, 3851-3854. proteolytic fragments missing the carboxyl-terminal end of 27. Carroll, S. F. & Collier, R. J. (1984) Proc. Natl. Acad. Sci. the S1 subunit do not bind the S2-S5 subunits of pertussis USA 81, 3307-3311. toxin. 28. Carroll, S. F. & Collier, R. J. (1987) J. Biol. Chem. 262, 8707- 8711. We thank Beatrice Aric6 for technical help and fruitful discus- 29. Tweten, R. K., Barbieri, J. T. & Collier, R. J. (1985) J. Biol. sions. Furthermore, we thank Marialuisa Melli, Roy Gross, and Chem. 260, 10392-10394. Giulio Ratti for critically reading the manuscript, Giorgio Corsi for 30. Douglas, C. M. & Collier, R. J. (1987) J. Bacteriol. 169, 4967- the drawings, and Susan Weemys and Angela 4971. Frazao for typing the 31. Allured, V. S., Collier, R. J., Carroll, S. F. & McKay, D. B. paper. This work was supported by a grant from Eute Nazionale (1986) Proc. Natl. Acad. Sci. USA 83, 1320-1324. Ideocazburi (ENI). 32. Nicosia, A., Bartoloni, A., Perugini, M. & Rappuoli, R. (1987) Infect. Immun. 55, 963-967. 1. Richter, C. (1987) in ADP-Ribosylation ofProteins: Molecular 33. Maniatis, T.. Fritsch, E. F. & Sambrook, J. (1982) Molecular Biology, Biochemistry and Biophysics, eds. Althaus, F. 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