Molecular Microbiology (1988) 2(1), 19-30
The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coii p. Glaser\ D. Ladant^ 0. Sezer', F. Pichot^ A. Ullmann^ and A. Danchin'* ^Regulation de I'Expression Genetique,, ^Bloehimle des Regulations Cellulaires, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Summary The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any S. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies. Introduction Bacterial virulence is a complex process involving intricate interactions between the prokaryotic organism and its eukaryotic host. Many pathogens secrete toxins that enter animal cells and impair their metabolism and function. The mechanisms of action of different toxins are far from being understood. It has been established that a specific class of protein toxins act in mammalian cells by elevating cyclic adenosine 3'5' monophosphate (cAMP) concentration: this leads to the alteration of several functions such as macrophage migration, phagocytic response and, more generally, proper utilization of cellular energy stores (Middlebrook and Dorland, 1984). In most cases, this is achieved by activation of the eukaryotic adenylate cyclases, by ADP-ribosylation of one of their regulatory subunits (Foster and Kinney, 1985). However, two pathogens, Bordetella pertussis and Bacillus anthracis (the aetiologic agents of whooping cough and anthrax, respectively), secrete, among virulence factors, an adenylate cyclase (Hewlett ef al., 1976; Leppla, 1982). The exact role of these adenylate cyclases in pathogenesis has not been elucidated, mainly because their characterization has Received 30 September, 1987. 'For correspondence.
proved extremely difficult, as has identification of the corresponding genes. For B. pertussis, however, it has been suggested that the adenylate cyclase enters animal cells and elicits an unregulated increase in intracellular cAMP synthesis, thereby disrupting normal cellular functions (Confer and Eaton, 1982; Friedman ef al., 1987). More importantly, 8. pertussis mutants deficient in adenylate cyclase have been shown to be avirulent (Weiss ef al., 1984). Adenylate cyclases have been the subject of intensive investigation because of their fundamental importance to most organisms. Although many aspects of their regulatory interactions have been described (Gilman. 1984), information about the structure of the catalytic subunits or domains is scarce, both for prokaryotic and eukaryotic enzymes. Determination of the cognate cya gene sequences from Escherichia eoli (Aiba ef al.. 1984) and Saeeharomyees eerevisiae (Kataoka etal.. 1985; Masson et al., 1986) has shown that the proteins have no obvious relationship, at least at the primary sequence level. It is therefore important to collect further information on these enzymes. A major feature of adenylate cyclase toxins is that they are strongly activated by calmodulin, a eukaryotic regulatory protein not known to occur in bacteria (Wolff et al., 1960; Masure et al., 1987). This remarkable interaction between proteins of phylogenetically very distant organisms makes a major contribution to virulence. Furthermore, it probably explains the difficulties encountered in the cloning of the genes. In the absence of caimodulin, the level of activity of the adenytate cyclase would be extremely low, if not zero, thereby precluding screening of recombinant clones by their cAMP production. We anticipated that the calmodulin regulation could be explored in the cloning of the S. pertussis adenylate cyclase gene. A cya-defective E. coli strain that expresses calmodulin was obtained. It was found to be a good recipient for revealing the cryptic adenylate cyclase activity of the toxin, thus permitting direct identification of the toxin gene. This is the first example of a general cloning procedure for genes whose products interact in a multicomponent protein complex.
Results Cloning of the B. pertussis adenylate cyclase in E. coli Several adenylate cyclase genes have been cloned in E eoli or in S. eerevisiae by screening complementing
20
P. Glaser, D. Ladant, O. Sezer. F. Pichot, A. Ullmann and A. Danchin
cyclase-defective clones on selective media (Wang etal.. 1981; Roy and Danchin, 1982; Hedegaard and Danchin, 1985; Kataoka ef a/.. 1985; Masson ef a/., 1986). Attempts to clone the S. pertussis adenylate cyclase using the same screening procedure failed, and no functional clones have been obtained. Various cloning vectors were used, which should have overcome possibilities of toxicity and tack of expression of the protein due to the absence of a transacting positive effector, as proposed by Weiss and Faikow (1984). These still yielded negative results. We assumed, therefore, that S. pertussis adenylate cyclase was not functional (or was poorly functional) in E. coli because it lacked the appropriate effector, namely calmodulin. We chose to use an E coli cya strain harbouring a plasmid, pVUC-1, which expressed high levels (50 p.gmg"' protein) of a synthetic calmodulin as a recipient for a library of totai B. pertussis DNA, constructed in a compatible replicon, pACYCi84 (Chang and Cohen, 1978). This calmodulin displays the characteristic features of calmodulin from higher vertebrates (Roberts e( al., 1985). Several clones complementing the E. coli cya defects were found. The corresponding plasmid DNA was then transformed into the parental E. coli strain lacking the pVUC-1 plasmid. •
•
n
•
A
The restriction map of some of the clones which express the B. pertussis enzyme, or only the catalytic part, are displayed in Fig. 1. The localization of the gene was obtained by cloning DNA fragments from B. pertussis using different restriction enzymes (see Experimental procedures). Two overlapping fragments, an 11 kb EcoHl fragment (1-8; pDIA6) and an 8.7 kb BamHI fragment (4-12: pDlA7 and pDIA8), permit synthesis of a calmodulin-sensitive adenylate cyciase. Plasmid pDIA4 analysis allowed a further narrowing of the cyclase window: activity is coded by a 2.1 kb region situated between SamHI site 4 and BgiU site 6. Estimation of the likely transcription orientation is provided by the study of the adenylate
Bam H 1 Bgl II Eco R 1 Eco RV Sma 1
?
p DIA 4
4
*
p DIA 5
J
2
4
5
T T TT 2
J
6
Tc 67
A 5
6 7 8
4
6 7 8
T T TT
p DIA 6
5
1 Cm
C
W t TTT
7 Cm"
o p DIA
5
A\ 1
p DIA
Under these conditions, there was no restoration of the positive phenotype on MacConkey-maltose plates, and no cAMP synthesis was measured in vivo. Thus, calmodutin expressed in £. eoli activates the 6. pertussis adenylate cyclase: this provided an explanation for the apparently general failure to directly clone the S. pertussis cya gene. This represents the first example of gene cloning with the aid of a protein effector from a heterologous organism, and is particular interesting in view of the phylogenetic divergence of the organisms involved.
8
Tc' 4
5
6 7 B
9
!T1T t • cya O R F •
1kb
R Tc
R Cm
Fig. 1. Restriclion maps of plasmids encoding the cya lcx;us ol B. pertussis. All plasmids are derivatives of pACYCI 84 (Chang and Cohen. 1978). Restriction sites are ordered following cya transcription orientation, Plasmids pDIA4.5,6 permit complementation of a eya strain of E. coli harbouring a plasmid. pVUC-1 (RobertsefaA, 1985) directing synthesis of calmodulin. In acalmodulin-free background, complementation is no longer visible. Plasmids pDIA7 and 8 could not be transformed inio E. coli synthesizing calmodulin, but they directed synthesis of 9. pertussis adenylate cyclase in a calmodulin-free background. Open lines represent the pACYCI 84 vector, solid lines the cya region, and the thin line represents, in pDIA4, an extra Saullla fragment (c. 100bp);inpDIA5the broken line (c. 7 kbp) stands for a B. pertussis DNA fragment that has not been identified as being actually present upstream from (he cya gene.
Bordetella pertussis adenytate cyctase cyclase synthesized from SamHl fragment 4-12, cloned into the unique BamHI site of plasmid pACYC184 (Chang and Cohen, 1978), This fragment was inserted in two orientations (pDIA7 and pDIA8) with respect to tetracycline gene transcription orientation, Adenylate cyclase synthesis differed widely in the two plasmids (see below, Table 1), suggesting that transcription proceeds from site 4 to site 12. Expression of the B. pertussis adenylate cyctase gene from pDIA8 demonstrated that, at least in E. coti. the presence of a positive transcriptional (or translational) effector is not an absolute requirement. Table 1. cAMP synthesis and adenylate cyclase activity ot recombinant clones.
Transforming pla5mid(s)
cAMP nmoles mg"'dry weight bacteria
Adenyjate cyciase aclivity nmoiesmin ' mg ' protein + CaM -CaM
pVUC-IpACYC184 pVUC-1pDIA4 pVUC-1pDIA5 pVUC-1pDiA6 pDIA7 pDIAS
< 0.001 15 43 38 0,01 < 0,001
< 0,01 0,36 0,60 0,25 0,60 0,07
lTyrAiggLuGlyWBl GGGGAACGTTTCAACGTGCGCAAGCAGTTGAACAACGCCAACGTGTATCGCGAAGGCGTG
974
2900 AlaThrGlnThrThcAlaTytGlyLysArgThrGluAsnWBlGlnTycArgHisValGlu GCTACCCAGACAACCGCCTACGGCAAGCGCACGGAGAATGTCCAATACCGCCATOTCGAG LauAlaArgValGlyGlnValValGluValAapThrLeuGluHiBValGlnMABll^llB CTGGCCCGTGTCGGGCAAGTGGTGGAGGTCGACACGCTCGAGCATGTGCAGCACATCATC 3000 , . . . y y y p y GGCGGGGCCGGCAACGA TTCG ATCACCGGCAATGCGCACGACAACTTCCTAGCC GGCOGQ
3100 SBrGlyAapAspAcgLouAspGlyGlyAlBGlyABnABpThrLeuVtlGlyGlyGluCly TCGGGCGACGACAGGCTGGATGGCGGCGCCGGCAACGACACCCTCGTTGGCGGCGAGGGC GlnABnThrVallleGlyGlyAlaGlyABpAspValPhaLeuGlnABpLBuGlyValTrp CAAAACACGGTCATCGGCGGCGCCGGCGACGACGTATTCCTGCAGGACCTGGGGGTATGG 3200 SerAsnGlnLBuAspGlyGlyAlaGlyValAapThcValLyaTyrAanValHiBGlnPro AGCAACCAGCTCGATGGCGGCGCGGGCGTCGATACCGTGAAGTACAACGTGCACCAGCCT SBrGluGluArgLeuGluArgHetGlyAspThcGlyllaHiBAlaABpLeuGlnLyBGly TC CG A GGA GC GCCTC G A AG GC A TGGGC G AC A CGG GC r«TCC A TGC CG A TCTTC A A A A GG GC 3300 . , . . ThrV»lGlui,yarcpPcoAlaL«uA»nL«uPhBS«rValAapHlBWalLy«ABnll»Glu ACGGTCGAGAAGTGGCCGGCCCTGAACCTGTTCAGCGTCGACCATGTCAAGAATATCGAG 3400
994
24
P. Glaser. D. Ladant, O. Sezer, F. Pichot, A. Uilmann and A. Danchin ATCTGC«COGCTCCCGCCT**»CO»CCGCATCGCCGGCaACGACC»GG*C»*CG*CCTC
LcuKBpGlyGIyAapGl.yArgABpThrValAspPna5arClyProGlyArgGlyl.auAap CTCGACGGCGGCGACGGCCGCGATACCGTGGATTTCAGCGGCCCGGGCCGGGGCCTCGAC
p y p y p g v TCOGGCCACGATGCCAACGACACGATACGCGGCCGGGaCCGCGACGACATCCTGCGCGGC
AlaGlyAlaUysGlyValPhELsuSarL-euGlyLysGlyPhaAlaSflrLauHalAapGlu GCCGGCGCAHAGGGCGTHTTCCTGAGCTTGGGCAAGGGGTTCGCCAGCCTGHTGGACGAA
3500
spVal1..*u GTGCTGATCGGCGACGCAGGCSCCAACGTCCTCAATGGCCTGGCGGGCAACGACGTGCTG
noo
3900
.
4600
GluTnrValSarAspAspllcAspGlyGlyAldGlyLauAapThrValAspTyrSerAla GAGACCGTCAGCGATGHCATCGUCGGCGGCGCGGQGCTCGHCACCGTCGACTACTCCOCC JfiOO . . . . MctllsHlBpToGLyAtglleValAlaPraHisGIuTyrGlyPheGlylieGluAlaHsp ATG*TCCATCCAGGCAGGATCGTTGCGCCGC»TGAATACGGCTTCGGG*TCGAGGCGGAC
D
.
ProGluTtirSBriiBtiValLauAcQAanllaGluAanAlaValGlySarAlaAigABpAap CCCGAAACCAGCAACGTGTTGCGCA*TATCG»GAACGCCGTGGGCAGCGCOCGTG*TGAC
TGCAGGACG*C
GGCCTGGGCCTCOJICACCCTGT*TGGCGAGG»CGGCA
1434 1454
IlallaHii! UaHlaAanGlnAlaVatAapGlnAlaGlyllaGluLya'LauifalCluAl* ATCATCCAISCCGCCAACCAGGCGGTAGACCAGGCAGGCATCGAAAA GCTGGTCGA GQC * . . SOOO HHitAlaGlnTyrProAspPtoGlyAlaAlaAlaMaAlaPcoPtoAlaAlaAcgValPro *TGGCGCAGTATCCQGACCCCGGCGCGGCGGCGGCTGCCCCGCCOGCCOCGCeCGTOCCC
167*
AapThrUauHeCGlnSecLsuAlaValAanTcpArg'** GACACGCTGATGCAG TCCCTGGCTGTC AACTGGCGCTGAAGCaCCGTGAATCACGGCCCG 5100 . . . . CCTGCCTCGCGCGGCGGCGCCGTCTCTTTGC GTTCTTCTCC G AG GT » TTTCCC ATC A TG A 5200 CGTCGCCC GTGGC GCAATGCGCCAGCGTGCCCGATTCCGGGTTGCTCTGCCTGGTCATGC
170fi
1B94
TGGCTCGCTATCACGGATTGGCAGCCGATCCCGAGCAGTTGCGGCATGdGTTCGCCGAGC 5100 A G GC A TTCTGT AGCGAAdCGATACAGCCTGGCGGCGCGCCGGGTCGCCCTGAAAGTOCGG CGGCACCGACCCGCGCCGGCGCGGCTGCCACCCGCGCCGCTGCCGGCCATCCCOCTGOdC S400 . . . . CGGCAGQGC GCCTACTTTGTT
i.A^A
Fig. 4. Nucleotide and deduced amino acid sequence of Ihe cya locus ot B. pertussis. The nucleotide sequence represents Ihe coding strand. Numbering of nudeolides starts at ttie A residue of Ihe ATG codon proposed as Ihe translation start of the protein. Two overlapping promoters matching Ihe E. coli consensus sequences (-35: TTGACA, - 10; TATAAT. and transcription start CAT) are indicated by boxes and brackets. The translation slart site has been determined according to criteria defined in the text: the doubly underlined nucleotides are complementary to the3'0H end of £.co/il6S RNA and might act as HBS Two protein domains, Band D. corresponding, as described in Fig. 5. to highly repetitive amino acid sequences are boxed. These sequence data have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00545.
pertussis toxin (Locht and Keith, 1986). A noteworthy feature of the codon usage, which was already prominent for tbe pertussis toxin genes, is the lack of symmetry: for instance, TCG or TAT are frequent codons, whereas their complements, CGA or ATA, are rare. In addition, there is a strong bias against codons ending with A or T. This is very different from the E. coli codon usage. It should be noted, however, that as in £. coli, CTG is the most frequent codon for leucine. These features could be used for assigning ORFs to unknown DNA sequences of B. pertussis: this would be very helpful in view of the fact that a high GC content excludes the presence of frequent translation termination codons. The overall amino acid content of the protein coded by the ORF (M, 177312) does not display any striking features, apart from the absence of cysteine residues and the high content of alanine, glycine, aspartate and asparagine. The absence of cysteine explains the observed resistance of the toxin to W-ethylmaleimide (Friedman ef ai.. 1987). There are no long stretches of hydrophobic amino acids, and the overall amino acid composition of
the protein is not hydrophobic, except perhaps for a signalsequence-like structure 400 amino acids downstream from the N-terminus (see below). Comparison of the protein sequence with itself revealed the existence of four regions or domains (Fig. 5). A first domain, present in pDIA4, of c. 400 amino acids, would correspond to the calmodulin-sensitive catalytic centre. This is followed by a domain of roughly 300 amino acids, very rich in alanine and glycine, and possessing an internal repetition of 13 amino acids (Fig. 5). A sequence of 250 amino acids follows, which has no obvious characteristic features, and the last 700 amino acids constitute a highly repetitive structure, made up of five repetitions of a structure which itself contains an internal glycine/aspartate-rich region repeated 7 to 9 times (see Fig, 5). A 13-amino-acidlong stretch present at the end of the third repetition of glycine repeats is also dearly reminiscent of the 13-aminoacid repeat present in domain B (Fig. 5, middle panel). These sequences have been used to screen protein data banks, and the only clear homology which was apparent was glycine-rich stretches present in several keratin
Bordetella pertussis adenyiate cyclase Table 2. Comparison ot codon usage between the 8. pertussis adenylate cyclase gene (upper figures) and the five pertussis toxin subunit genes (lower figures). F TTT F TTC L TTA L TTG
2 3 40 25 0 1 21
S TCT
CTT
L CTC L CTA
8 7 18 24 3
TAT
Y
TAC
•
TAA
•
TAG
0 1
2 3 10 17 3 3 22 2B
H
CAT
18 11 16 11 14 9 56 24
3 10 40 35 5 5
N AAT
1
S
TCC
s
TCA
s
TCG
23 23 1 2 32 9
It L
Y
3
p
CCT
p
CCC
p
CCA
1
H CAC
a
CAA
Q CAG
16 28 25 35 0 0
C TGT C TGC •
TGA
w
TGG
R CGT
9 6
R CGC
59 33 2 1 22 13
n
CGA
R CGG
L
CTG
96 48
p
CCG
1
ATT
11 9
T
ACT
1
ATC
57
T
ACC
ATA
31 1 7
T
ACA
M ATG
22
T
ACG
31 27
K AAG
32 24
R AGG
A
GCT
D GAT
GCC
A
GCA
87 33
E GAG
56 9 104 29 34 29 50 15
G GGT
A
13 59 112 52 14 12
1
4
V GTT V GTC V GTA V GTG
5 5 45 33 8 7 77 17
A GCG
N AAC K AAA
D GAC E GAA
19 B 49 20 11 4
0 0 0 25 1 4 15 10
s
AGT
2 1
s
AGC
37 36 0 4 4 7
R AGA
G GGC G GGA G GGG
16 5 176 59 12 12 35
25
a signal permitting secretion of the cyclase. U should be noted that several secretion signals occur internally, such as the ovalbumin signal present after the first third of the protein (Lingappa ef ai., 1979) or the opsin signal sequences that are intemal to the protein (Friedlander and Blobel, 1985). No homology could be found either with the other known bacterial cyclase (Aiba ef ai., 1984) or with yeast cyclase (Kataoka ef ai., 1985; Masson ef ai., 1986). The only feature in common with the E coii equivalent protein (848 amino acids) is the presence of a catalytic domain of c. 400 amino acids at the NHs-terminus of the protein (Roy ef al., 1983); this contrasts with the S. eerevisiae enzyme (2026 amino acids), in which the catalytic domain of c. 400 amino acids is carboxy-terminal (Kataoka et ai, 1985; Masson ef ai., 1986). However, as in the yeast enzyme, a highly repetitive sequence was observed, although amino-terminal in yeast and from a completely different motif. This was somewhat surprising and might suggest several pathways for convergent evolution in cAMP synthesis. Homologies with other proteins have been looked for in polypeptide banks (PSeqIP and NBRF) and some homology has been discerned between the first 450 amino acids and nucleotide binding proteins such as dehydrogenases. For instance, a significant homology of 30%, if one takes into account amino acids from the same class (Schwartz and Dayhoff, 1978) was found with E. coii IMP dehydrogenase.
4
The cya gene comprises 1707 codons (including tbe terminal TGA cotJon) whereas tbe sum of pertussis toxin subunits comprises 977 codons Despite the significant difference in amino acid compositian of tbe proleins, it is clear thai the codon usage is very similar in both sets of genes.
genes. This might suggest a filamentous {HoffrTian et ai.. 1985), or periodical (Green and Warren, 1985) structure for the cyclase precursor. Although calmodulin sites are notoriously difficult to identify, the sequence DLLWKIARAGARSAVGTEK displays slight homology with sequence KRRWKKNFIAVSAANRFKKI of skeletal muscle myosin light-chain kinase or RRKWQKTGHAVRAIGRLSSS of smooth muscle myosin light-chain kinase (O'Neil et al.. 1987) and we propose that it corresponds to a peptide interacting with calmodulin. The repetitions present in the domain located downstream from the catalytic domaifi are clustered in two places, bordering polypeptides of 620 and 690 amino acids. This matches the size of the two protein bands present when the B. pertussis cya gene is expressed in E. coii (see Fig. 3). In addition, the corresponding consensus sequence, ASAAAGAVAAA-G, is very similar to the core of signal peptides of proteins exported by £. coli (Gascuel and Danchin, 1986). This might correspond to
Discussion S. pertussis adenylate cyclase is of interest for several reasons: it is a toxin and therefore released extracellularly; it has the highest specific activity reported for any adenylate cyciase (1600 j^mol of cAMP min ^ mg \ Ladant ef ai., 1986); and it is activated by calmodulin. These unusual features raise questions as to the regulation of the activity and evolution of this enzyme. The molecular analysis of the corresponding gene was. therefore, the first step towards answering some of these questions Controversial data on the molecular weight of the enzyme may be relevant to its processing and secretion mechanisms. The purified, extracellular enzyme has been reported to have a molecular weight of 70 kD (Hewlett and WolH. 1976) and, more recently, of 43-50 kD (Shattuck e( at., 1985: Ladant ef ai.. 1986). High-molecular-weight forms of the enzyme were isolated from culture supernatants (Kessin and Franke, 1986). The molecular weight of unaggregated invasive' forms of B. pertussis adenylate cyclase was around 190 kD (Hanski and Fartel. 1985). In addition, we showed that antibodies raised against the 43-50 kD form of the enzyme react in Western blots of virulent S. pertussis extracts with several polypeptides whose molecular weights range between 45 and 200 kD
26
P. Glaser, D. Ladant, 0. Sezer, F. Pictiot, A. Ullmann and A. Danchin
GLGAAPGVPSGRS ASPGLRRPSLGAV VSDMAAVEAAELE A^L QGAQAVAAAQR AASLSAAVFGLGE SSAVAeTVSGFFR 5SRMAGGFGVAGG AMALGGGIAAAVG PDAPAGQKAAAGA ASSIAL.ALAAARG AGAAAGALAAALS SSAYGYEGDALLA KTAAEGAVAGVSA TVGAAVSIAAAAS TGAHAGIAAGRIG
D
ASAAAGAVAAA-G
913 GGDGpDVVLANASRIHYD p 1015 102 4 103 3 104 2 1051 1060 1080 1138 1147 1155 1165 1174 1183 120 3 1262 1271 1280 1289 1298 130 7 1316 1325 134 5 1403 I'll2 1421 14 30 14 39 1448 1457 1477 1529 15 38 154 7 1556 1565 15 74 1583 1604
GGA^TNTVSYAALGRQDSITVSADGERFNVRKQLNNANVYREGVATQTTAYGKRTENVQ GGAGNDSIT GGSGDDRL.D GGAGNDTLV GGEGQNTVI GGAGDDVFLQDLGVWSNQLD GGAGVpTyKYNVHQPSEERLERMGDTGIHADLQKGTVEKHEPALNLFSVDHVKNIENLH GSRLNDRI^A GDDQDNELW \ GHDGNDTIR GRGGGDDILR GGLGLDTLY GEDGNDIFLQDDETVSDDID GGAGLDTVDYSAHIHPGRIVAPHEYGFGIEADLSREWVRKASALGVDYYDNVRNVENVI GTSMKDVLI GDAQANTLH GQGGDDTVR GGDGDDLLF GGDGNDMLY GDAGNDTLY GGLGDDTLE GGAGNDWFGQTQAREHDVLR GGDGVDTVDYSQTGAHAGIAAGRIG_LGILADLGAGRVDKLGEAGSSAYDTVSGIENVV GTELADRIT GD^QA^VLR GAGGADVLA GGEGDDVLL GGDGDDQLS GDAGRDRLY GEAGDDWFFQDAANAGNLLD GGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLHDEPET5NVLRNIENAV GSARDDVLI GDAGANVLN GLAGNDVLS GGAGDDVLL GDEGSDLLS GDAGNDDLF GGQGDDTYLFGVGYGHDTIYE SGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIH
(GGAGDDTL-) '5-7 GGAGDD-FLQD-A-~SD-LD GGAGDVTVDYS(-) , . , G - G I - A D L ( - ) ^ K ( - ) ^ D - V - N - E N V -
consensus
Consensus
Fig, 5. Comparison ol the adenylale cyclase polypeptide with itself. The amino acid sequence ol adenylate cyclase, deduced from the nucleotide sequence (one tetter coda), was compared wilh itself. Stretches ol 15 amino acids having 5 amino acids in common were sought Matching stretches are represented by a diagonal line. Four main domains (upper panel) are revealed by the presence of internal repeats. Domain A (c, 400 amino acids) corresponds !o the catalytic centre of the protein. Domain B (c. 300 amino acids), which can be split in two parts limited by alanlne-rich repeats (middle panel), links the catalytic centre to domain C, which does not display significant repetition. Finally, the lasl 700 amino acids constitute a highly periodical structure (D) consisting of five repeats each containing 7 to 9 glycine-rrch repeats (lower panel). Consensus sequences are determined using identical amino acids (bold letters), and amino acids from Ihe same class (underlined letters), according to Schwartz and Dayhofl (1978),
Bordelella pertussis adenylate cyclase (unpublished results). Our sequence data showed that the gene encodes a polypeptide of 1706 amino acids. Thus, the unprocessed and the non-secreted protein has a much higher molecular weight than the purified enzyme. Our immunochemical results showed that adenylate cyclase expressed in £. coti exhibits a significantly higher molecular weight (between 80 and 120 kD) than the purified extracellular enzyme (Fig, 3). It seems, therefore, that the high-molecular-weight gene product of the S, pertussis cya gene is degraded at its carboxy-terminal end to give a form that retains its ability to be activated by calmodulin. Calmodulin activation is a unique feature of the adenylate cyclase toxins. It is also one of their most intriguing aspects. We have demonstrated that we could reconstitute in vivo the active form of 8, pertussis calmodulin-dependent adenylate cyclase. This was done in a heterologous system involving three partners (a eukaryotic protein, calmodulin, a prokaryotic protein, the B. pertussis adenylate cyclase, and E coti) that allowed the simultaneous synthesis of the two proteins. This was of particular interest since more straightforward reconstitution experiments involving partners of homologous origin, such as S, cerevisiae adenylate cyclase and its presumed RAS protein activator, have led. for instance, to conflicting observations. Uno ef at. (1985) claimed that they could reproduce in E coti the activation of the adenylate cyclase, whereas Kataoka ef at. (1985) found that they failed to reproduce the same activation. Calcium is not an absolute requirement for activation of S, pertussis adenylate cyclase (Greenlee et al., 1982). This contrasts with other mammalian calmodulin-sensitive enzymes, including adenylate cyclases such as the rat brain enzyme. This less stringent interaction may have some relevance to the evolution of calmodulin binding domains. From studies on calmodulin binding sites of mammalian calmodulin-dependent protein kinases, no consensus sequence has been defined (Bennett and Kennedy, 1987). Nevertheless, the existence of an immunological relationship between rat brain and S. pertussis adenylate cyclases argues in favour of a common evolutionary origin of these calmodulin-sensitive enzymes (Monneron et at., unpublished data). We have determined the nucleotide sequence of a single clone carrying the cya locus of B. pertussis. The determined sequence displays one large open reading frame with the potential to encode a very large protein (1706 or 1740 codons, according to the chosen translation start site). Analysis of the predicted amino acid sequence of the protein revealed several striking features. First of all. there do not seem to be the long hydrophobic stretches expected for a protein that is secreted and whose catalytic domain seems to have strong affinity for membranes (Ladant ef al., 1986). Other microbiai adenylate cyclases are primarily membrane-associated (e.g. the E ooli or the
27
S. cerevisiae adenylate cyclases) or cytoplasmic (e.g. the enzymes from Brevibacterium ilquefaciens or Staphytccccous salvarius] (Ide. 1971)). As in the case of E coil and S. oerevisiae adenylate cyclases (Aiba et at., 1964; Kataoka ef al., 1985), membrane localization of B. pertussis adenylate cyclase does not seem to be related to the amino acid composition of the protein, but, rather to some unknown structural feature of the secreted polypeptide. It is worth noting that no signal-peptide-like sequence could be translated from the 5' end of the ORF, such as would be expected for a secreted protein Several sequences with homology to a consensus signal-peptide sequence that might be Involved in secretion, are present downstream from the amino-proximal 400 amino acids. Finally, we find that a carboxy-terminal domain of 700 amino acids consists of repetitions of a glycine-rich motif. This might give periodical or filamentous properties to the protein, and reveal a hydrophobic character, permitting, for instance, exposition of the catalytic centre on the external surface of the cell. A processing system might result in toxin secretion in B. pertussis. This system did not appear to be present in E coli. One is tempted to speculate on another function of the large fragment which would then remain present on the bactenal surface: thus it might have relevance to the haemolytic property of virulent B. pertussis. In this respect, it may be significant that, among Tn5-induced mutations affecting virulence in B. pertussis, Weiss ef al. (1984) have not obtained mutants lacking only adenylate cyclase. These authors obtained either haemotysin-deficient mutants or haemolysin-adenylatecyclase double mutants. If the 5.2 kb ORF encoded a multifunctional protein, the processing, activation and secretion might involve more complex interactions than previously thought. The mechanism for invasion of animal cells by B. pertussis adenylate cyclase has not been elucidated. It is likely that it contributes to the evasion of host defences by suppressing phagocytic functions (Confer and Eaton. 1982). The expression of the adenylate cyclase gene in E coli wiil allow the construction of different truncated genes to check whether the entire gene product or only part of it would enter animal cells. It is possible that adenylate cyclase, like other protein toxins, exhibits a catalytic activity and a function that enables the toxin to interact with the host cells (Middlebrook and Dorland, 1984). The high-molecular-weight gene product (177 kD) in compahson with the 43-50 kD active enzyme, would support such a hypothesis. The successful expression of S, pertussis adenylate cyciase in E coli suggests that it may be possible to apply our novel cloning strategy to a variety of other systems that play a key role in cellular regulation, particularly in caimodulin-sensitive regulatory enzymes, such as protein kinases, phosphodiesterases and adenylate cyclases.
28
P. Glaser. D. Ladant. O. Sezer. F. Pichot, A. Ullmann and A. Danchin
Experimental procedures Bacterial strains and growth media A cya derivative of the restriction-minus E. coti strain, C600SF8 (Struhl ef at., 1976). TP610 was used throughout this work (Hedegaard and Danchin, 1985), Bacteria were grown in rich medium, LB. or minimal medium, M63 (Miller, 1972). Screening for the ability to ferment sugars was performed on MacConkey agar plates containing 1 % of the sugar tested. Antibiotic concentrations were: tetracycline, 10 p.g m l " ' ; chloramphenicoi. 30 ^.g ml"'; ampicillin. 50 jig m\~\ Strain TP610 was transformed (Maniatis ef al., 1982) with plasmid pVUC-1 {a gift from Drs J. Haiech and D,M, Watterson) and checked for calmodulin production. This transformed strain was used as a recipient for cloning a pertussis DNA fragments (strain 18323 W.H.O,).
Screening procedures B. coti strain TP610pVUC-1 is resistant to ampicillin, and the plasmid is from the IncQ incompatibility group. Thus, we chose to clone 6. pertussis DNA in a compatible replicon, pACYC184 (Chang and Cohen, 1978), selecting for chloramphenicoi resistance. Sat/ilia partial digests of total B. pertussis DNA were ligated into pACYC184 DNA cut with SarnHL Clones were screened for maltose fermentation on MacConkey plates supplemented with 100 (i.g ml"' ampicillin, 20 fig m r ' chloramphenicoi. and 1 % maltose. Clones unable to synthesize cAMP cannot ferment maltose, and are white on such plates, whereas clones which produce cAMP ferment maltose and are deep red in colour. Red clones were picked up. purified on the same medium, and checked for p-galactosidase synthesis on LB plates supplemented with the chromogenic p-galactosidase substrate, Xgat. and the lactose operon inducer, IPTG {M'\\\er. 1972); cAMP-deficient bacteria are unable to express the lactose operon. The plasmids were analysed by restriction enzyme digestion and further transformed, either into TP610, where they remained cya-negative, or back into TP610pVUC-1. where cAMP synthesis was restored. When preliminary restriction maps had been obtained, other 6, pertussis DNA fragments were cloned directly into pACYC184. after total restriction with either SamHl or EcoRI, The selection used in the case of EccRI restriction was tetracycljne (10 \xg ml'') because the unique EcoRI site of pACYC184 interrupts the chloramphenicoi resistance gene. For unknown reasons, SamHl cloning always simultaneously yielded several fragments, including the 8,7 kb fragment 4-12 that encompassed the total cya gene. It was possible, however, to clone this fragment alone, albeit in strain TP610 devoid of plasmid pVUC-1. The reason for the calmodulin-induced toxictty is not yet understood.
DNA sequence analysis DNA sequence analysis was performed using subclones in tha phage M13tg131 (Kieny ef ai.. 1983), Unidirectional deletions were generated using the Cyclone System (IBI), The DNA sequence was determined by the dideoxynucleotide method of Sanger e( at. (1977), using {alphaS^^)dATP (Amersham) and 7deaza-dGTP (Boehringer) instead of dGTP, since this results in better resolution of GC-rich regions in polyacrylamide gels (Mizusawa ef at.. 1986), Sequence analysis was performed using
the facilities of the Unitd d'Informatique Scientifiqua of the Pasteur Institute-
Determination of cAMP concentrations Bacteria from an overnight cultura grown In LB medium in the presence of antibiotics were diluted in M63 and heated for 5 min at 100X, Total cAMP was assayed using a standard radioimmmunoassay (Joseph ef at., 1982).
Assay of adenytate cyciase Bacteria from an overnight culture grown in LB medium in the presence of antibiotics were centrifuged, washed with M63 medium, and sonically disrupted in Tris-HCl 20 mM, MgCI? 10 mM, pH 8, After centrifugation, adenylate cyciase was assayed in the supernatants, as described by Ladant ef a/. (1986). The reaction was performed at 30X in a volume of 100 M.1 containing 50 mM Tris-HCl (pH 8) 2 mM (alpha'^P}ATP (5x10^ cpm/assay) 6 mM MgCl2. 100 ii.g bovine serum albumin. 0,13 mM (^H)cAMP (1.5x10" cpm/assay). 0,12 mM CaClj and 0,1 M-M calmoduiin (when added). One unit cf adenylate cyclase corresponds to 1 nmole of cAMP formed in 1 min at 30°C.
Purification and immunologioal detection of adenylate cyclase B. pertussis adenytafe cyclasa was purified from culture supernatants. as described by Ladant ef at. (1986)- Cloned S. pertussis adenylate cyclase was partially purified from E coti strain TP610pDIA7 as follows. An overnight culture in LB medium supplemented with chloramphenicol was centrifuged. and the bacteria were sonically disrupted in Tris HCl 30 mM pH 8, NaCI 15 mM and CaCl2 0,5 mM, 20 ml of the bacterial extract (140 units ml"' adenylate cyciase, 6 mg ml ' protein) were mixed with calmodulin-agarose (Sigma) packed gel and shaken at 4 X for 18 h. The gel which retained 50% of the enzymatic activity was centrifuged and washed with Tris HCl (60 mM) pH 8, NaCI (15 mM), CaClj (0.1 mM) and NP40 (0-1 %), Adenylate cyclase was eluted with the same buffer containing 8 M urea. The eluted adenyiate cyciase was diaiysed overnight against Tris HCl (50 mM) pH 8, CaCl2 (0,1 mM), NP40 (0,1%), For immunoblots. proteins separated by SDS-PAGE (10%) were transferred to nitrocellulose sheets and incubated with anti-8. pertuss/s adenylate cyclase polyctonal serum (dilution 1 /1000) obtained frcm guinea pigs. The detection was performed with '"i-iabelled protein A,
Notes added in proof We have found that the 1300 COOH distai amino acids of the protein are highly hcmoiogous to the tilyA gene product (haemoiysin) of E. coti (Felmiee ef at.. 1985; J Bad 163: 94-105). This suggests fhat both adenylate cyclase and haemoiytic activity are carried by the same protein in B, pertussis.
Acknowledgements We thank J, Haiech and D, M, Watterson for the gift of plasmid pVUC-1. O, Barzu for discussions on the nature of the protein, K, M, Kean for critical reading of the manuscript and for linguistic
Bordetella pertussis adenytate cyclase editing, and G. Limeul for expert typing, Financiai support came from the Centre Nationai de la Recherche Scientifique (UA 1129) and the institut Pasteur (3840 and 3845),
References Aiba, H., Mori, K.. Tanaka, M,, Oci, T., Roy, A., and Danchin, A. (1984) The complete nucleotide sequence of the adenylate cyclase gene of Escherichia coti. Nuct Acids Res 12; 94279440. Bennett, iH,K,, and Kennedy, M.B, (1987) Deduced primary structure of Ihe subunit of brain type II Ca^Vcalmodulin-dependent protein kinase determined by molecular cloning, Proc NatiAcad Sci USA 6A: 1794-1798, Chang, A.C, and Cohen, S.N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the PI 5A cryptic miniplasmid, J Bacteriot 134:1141 -1156, Confer, D,L., and Eaton, J,W, (1982) Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217: 948-950, Creighton, T,E, (1984) Proteins: Structures and Motecuiar Properties. San Francisco: W.H, Freeman, Deuschle, U,, Kammerer, W., Centz, R,, and Bujard, H, (1986) Promoters of Bsctierictila coti: a hierarchy of in vivo strength indicales alternate structures, EMBO J 5: 2987-2994, Farlei, Z,, Friedman, E,, and Hanski, E, (1987) The invasive adenylate cyclase of B. pertussis. Intraceilular localization and kinetics of penetration into various celis, Biochem J 243; 153158, Foster, J,W,, and Kinney, D.M. (1985) ADP-ribosylating microbiai toxins, Crit Rev Microbioi 11: 273-298. Friedlander, M,, and Blobel, G. (1985) Bovine opsin has more than one signal sequence. Nature 318; 338-343Friedman, E., Farfel, Z,, and Hanski, E. (1987) The invasive adenylate cyclase of B. pertussis. Properties and penetration kinetics, Biochem J 243; 145-151, Gascuel. O,, and Danchin, A. (1986) Prolein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation, J Mot Evot 24: 130-142. Gilman, A,G, (1984) G proteins and dual control of adenyiate cyclase, Cetl 36: 577-579. Green, R,L,, and Warren, G,J, (1985) Physical and functional repetition in a bacteriai ice nucieation gene. Nature 317: 645648, Greenlee, D.V,, Andreasen, T,J,, and Storm, D.R. (1982) Calciumindependent stimulation of Bordetetta pertussis adenylate cyclase by oalmodulin. Biochemistry 21; 2759-2764, iHanski, E., and Farfel, Z. (1985) Bordetetta pertussis invasive adenyiate cyclase. Partial resolution and properties of its cellular penetration, J Biol Chem 290; 5526-5532, Hedegaard, L,, and Danchin, A. (1985) The cya gene region of Erwinia chrysanthemi B374: organisation and gene products. Mot Gen Genet 201: 38-42, Hewlett, EL,, and Wolff, J, (1976) Solubie adenyiate cyclase from the culture medium of Bordetelta pertussis: purification and characterization, J Bacteriot 127; 890-898, Hewiett, E,L., Urban, M.A., Manclark, C,R., and Wolff, J, (1976) Extracytoplasmic adenylate cyclase from Bordetetia pertussis. Proc Natt Acad Sci USA 73; 1926-1930, Hoffmann, W,, Franz, J,K,, and Franke, W,W. (1985) Amino acid sequence microheterogeneities of basic (type II) cytokeratins of Xenopus taevis epidermis and evolutionary conservativity of helical and non-helical domains, J Mol Biot 184: 713-724. Ide, M, (1971) Adenylate cyciase of bacteria. Aroh Biochem
Biophys ^M: 262-268.
29
Joseph, E., Bemsley, C , Guiso, N,, and Ullmann, A, (1982) Multiple reguiation ofthe activity of adenylate cyclase in Escherichia ooti. Mol Gen Genet 185: 262-268, Kataoka, T,, Broek, D,, and Wigler, M. (1985) DNA sequence and characterization of the S, cerevistae gene encoding adenyiate cyclase, Cetl 43; 493-505, Kessin, R,H,, and Franke, J, (1986) Secreted adenylate cyclase of Bordetetta pertussis: calmodulin requirements and partial purification of two forms, J Bacteriol 166; 290-296. Kieny, MP,, Lathe, R,, and Lecocq, J P . (1983) New versatiie cloning and sequencing vectors based on bacteriophage M13, Gene 26: 91-99. Ladant, D,, Brezin, C , Aionso, J,-M., Crenon, i,, and Guiso, N. (1986) Bordetelta pertussis adenylate cyciase Purification, characterization and radioimmunoassay, J Biol Chem 261: 16264-16269. Leppla, S,H, (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMF concentrations in eukaryotic celis, Proc Natl Acad Sci USA 79: 3162-3166. Lingappa, V.R., Lingappa, J.R., and Blobel, G, (1979) Chicken ovalbumin contains an internal signal sequence. Nature 281; 117-121Locht, C , and Keith, J-M, (1986) Pertussis toxin gene: nucleotide sequence and genetic organization. Science 232; 1258-1263. Maniatis, T., Fritsch, E.F,, and Sambrook, J, (1982) Molecular Ctoning: A Laboratory Manuat. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, Masure, H.R., Shattuck, R,L,, and Storm, D,R, (1987) Mechanisms of bacterial pathogenicity that involve production of calmodulin-sensitive adenyiate cyclases, Microbiot Rev 5 1 ; 60-65. Masson, P-. Lenzen, G,,Jacquemin, J.M,, and Danchin, A. (1986) Yeast adenylate cyclase catalytic domain is carboxy terminai, Curr Genet 10: 343-352, Miller, J.H. (1972) Experiments tn Molecular Genetics. Cold Spring Harbor, New York: Coid Spring Harbor Laboratory. Middiebrook, J,L,, and Dorland, R,B, (1984) Bacterial toxins: cellular mechanisms of action, Microbiot Rev 48; 199-221Mizusawa, S,, Nishimura, S,, and Seeia, F, (1986) Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP, NucI Acids Res 14; 1319-1324, Nicosia, A., and Rappuoli, R. (1987) Promoter of fhe pertussis toxin operon and production of pertussis toxin. J Bacteriol 169: 2843-2846 O'Neil, K.T,, Wolfe, H,R,. Jr,, Ehckson-Vitanen, S,, and DeGrado, W,F, (1987) Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity. Science 236: 14541456, Roberts, DM,, Crea, R,, Malecha, M,, Alvarado-Urbina, G,, Chiarello, R.H,, and Watterson, DM, (1985) Chemical synthesis and expression of a calmoduiin gene designed for site-specific mutagenesis. Biochemistry 24; 5090-5098, Roy, A,, and Danchin, A. (1982) The cya locus of Escherichia CO//K12: organization and gene products. Mot Gen GeneM88: 465-471. Roy, A,, Danchin, A,, Joseph, E,, and Ullmann, A, (1983) Two functional domains in adenylate cyciase of Escherichia coti. J Mot Biot 165: 197-202, Sanger. F,, Nicklen, S., and Couison, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc Natt Acad Sci USA 74; 5463-5467. Schwartz, RM,, and Dayhoff, M,0- (1978) In Atlas of Protein Sequence and Structure, vol 5. Dayhoff, M,O- (ed.) Washington, D.C: National Biomedical Research Foundation, pp, 353-358,
30
P. Glaser. D. Ladant, O. Sezer, F. Pichot, A. Ullmann and A. Danchin
Shattuck, R.L.. Oldenburg, D.J.. and Storm, D.R, (1985) Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis. Biochemistry 24: 63586362. Shepherd, J.CW. (1981) Method to determine the reading frame of the protein from the purine/pyrtmidine genome sequence and its possible evolutionary justification. Proc NatI Acad Sci USA7S: 1596-1600. Struhl. K., Cameron. J.R., and Davis, R.W. (1976) Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc NatI Acad Sci USA 73: 1471-1475. Uno. I,, Mitsuzawa, H-, Matsumoto, K,, Tanaka, K., Oshima, T., and Ishikawa, T. (1985) Reconstitution ot the GPT-dependent adenylate cyclase from products of the yeast cyri and ras2 genes in Escherichia coli. Proc Natt Acad Sci USA 82: 78557859.
Wang, J.Y.L. Clegg, D.O., and Koshland, D.E., Jr. (1981) Molecular cloning and amplification of the adenylate cyclase gene. Proc NatI Acad Sci USA 78: 4684-4688. Weiss, A.A., and Falkow, S. (1984) Genetic analysis of phase change in Bordetella pertussis. Infect Immun 43: 263-269. Weiss, A.A., Hewlett, E.L., Myers, G.A. and Falkow. S. (1984) Pertussis toxin and extracytopiasmic adenylate cyclase as virulence factors in Bordetella pertussis. J Infect Dis 150:219222. Wolff, J., and Hope Cook, G. (1982) Amphiphile mediated activation of soluble adenylate cyclase of Bordetella pertussis. Arch Biochem Biophys 215: 524-531. Wolff, J., Hope Cook, G., Goldhammer, A.R., and Berkowitz, S.A. (1980) Calmodulin activates prokaryotic adenylate cyclase. Proc NatI Acad Sci USA 77: 3840-3844-
The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coii p. Glaser\ D. Ladant^ 0. Sezer', F. Pichot^ A. Ullmann^ and A. Danchin'* ^Regulation de I'Expression Genetique,, ^Bloehimle des Regulations Cellulaires, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Summary The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any S. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies. Introduction Bacterial virulence is a complex process involving intricate interactions between the prokaryotic organism and its eukaryotic host. Many pathogens secrete toxins that enter animal cells and impair their metabolism and function. The mechanisms of action of different toxins are far from being understood. It has been established that a specific class of protein toxins act in mammalian cells by elevating cyclic adenosine 3'5' monophosphate (cAMP) concentration: this leads to the alteration of several functions such as macrophage migration, phagocytic response and, more generally, proper utilization of cellular energy stores (Middlebrook and Dorland, 1984). In most cases, this is achieved by activation of the eukaryotic adenylate cyclases, by ADP-ribosylation of one of their regulatory subunits (Foster and Kinney, 1985). However, two pathogens, Bordetella pertussis and Bacillus anthracis (the aetiologic agents of whooping cough and anthrax, respectively), secrete, among virulence factors, an adenylate cyclase (Hewlett ef al., 1976; Leppla, 1982). The exact role of these adenylate cyclases in pathogenesis has not been elucidated, mainly because their characterization has Received 30 September, 1987. 'For correspondence.
proved extremely difficult, as has identification of the corresponding genes. For B. pertussis, however, it has been suggested that the adenylate cyclase enters animal cells and elicits an unregulated increase in intracellular cAMP synthesis, thereby disrupting normal cellular functions (Confer and Eaton, 1982; Friedman ef al., 1987). More importantly, 8. pertussis mutants deficient in adenylate cyclase have been shown to be avirulent (Weiss ef al., 1984). Adenylate cyclases have been the subject of intensive investigation because of their fundamental importance to most organisms. Although many aspects of their regulatory interactions have been described (Gilman. 1984), information about the structure of the catalytic subunits or domains is scarce, both for prokaryotic and eukaryotic enzymes. Determination of the cognate cya gene sequences from Escherichia eoli (Aiba ef al.. 1984) and Saeeharomyees eerevisiae (Kataoka etal.. 1985; Masson et al., 1986) has shown that the proteins have no obvious relationship, at least at the primary sequence level. It is therefore important to collect further information on these enzymes. A major feature of adenylate cyclase toxins is that they are strongly activated by calmodulin, a eukaryotic regulatory protein not known to occur in bacteria (Wolff et al., 1960; Masure et al., 1987). This remarkable interaction between proteins of phylogenetically very distant organisms makes a major contribution to virulence. Furthermore, it probably explains the difficulties encountered in the cloning of the genes. In the absence of caimodulin, the level of activity of the adenytate cyclase would be extremely low, if not zero, thereby precluding screening of recombinant clones by their cAMP production. We anticipated that the calmodulin regulation could be explored in the cloning of the S. pertussis adenylate cyclase gene. A cya-defective E. coli strain that expresses calmodulin was obtained. It was found to be a good recipient for revealing the cryptic adenylate cyclase activity of the toxin, thus permitting direct identification of the toxin gene. This is the first example of a general cloning procedure for genes whose products interact in a multicomponent protein complex.
Results Cloning of the B. pertussis adenylate cyclase in E. coli Several adenylate cyclase genes have been cloned in E eoli or in S. eerevisiae by screening complementing
20
P. Glaser, D. Ladant, O. Sezer. F. Pichot, A. Ullmann and A. Danchin
cyclase-defective clones on selective media (Wang etal.. 1981; Roy and Danchin, 1982; Hedegaard and Danchin, 1985; Kataoka ef a/.. 1985; Masson ef a/., 1986). Attempts to clone the S. pertussis adenylate cyclase using the same screening procedure failed, and no functional clones have been obtained. Various cloning vectors were used, which should have overcome possibilities of toxicity and tack of expression of the protein due to the absence of a transacting positive effector, as proposed by Weiss and Faikow (1984). These still yielded negative results. We assumed, therefore, that S. pertussis adenylate cyclase was not functional (or was poorly functional) in E. coli because it lacked the appropriate effector, namely calmodulin. We chose to use an E coli cya strain harbouring a plasmid, pVUC-1, which expressed high levels (50 p.gmg"' protein) of a synthetic calmodulin as a recipient for a library of totai B. pertussis DNA, constructed in a compatible replicon, pACYCi84 (Chang and Cohen, 1978). This calmodulin displays the characteristic features of calmodulin from higher vertebrates (Roberts e( al., 1985). Several clones complementing the E. coli cya defects were found. The corresponding plasmid DNA was then transformed into the parental E. coli strain lacking the pVUC-1 plasmid. •
•
n
•
A
The restriction map of some of the clones which express the B. pertussis enzyme, or only the catalytic part, are displayed in Fig. 1. The localization of the gene was obtained by cloning DNA fragments from B. pertussis using different restriction enzymes (see Experimental procedures). Two overlapping fragments, an 11 kb EcoHl fragment (1-8; pDIA6) and an 8.7 kb BamHI fragment (4-12: pDlA7 and pDIA8), permit synthesis of a calmodulin-sensitive adenylate cyciase. Plasmid pDIA4 analysis allowed a further narrowing of the cyclase window: activity is coded by a 2.1 kb region situated between SamHI site 4 and BgiU site 6. Estimation of the likely transcription orientation is provided by the study of the adenylate
Bam H 1 Bgl II Eco R 1 Eco RV Sma 1
?
p DIA 4
4
*
p DIA 5
J
2
4
5
T T TT 2
J
6
Tc 67
A 5
6 7 8
4
6 7 8
T T TT
p DIA 6
5
1 Cm
C
W t TTT
7 Cm"
o p DIA
5
A\ 1
p DIA
Under these conditions, there was no restoration of the positive phenotype on MacConkey-maltose plates, and no cAMP synthesis was measured in vivo. Thus, calmodutin expressed in £. eoli activates the 6. pertussis adenylate cyclase: this provided an explanation for the apparently general failure to directly clone the S. pertussis cya gene. This represents the first example of gene cloning with the aid of a protein effector from a heterologous organism, and is particular interesting in view of the phylogenetic divergence of the organisms involved.
8
Tc' 4
5
6 7 B
9
!T1T t • cya O R F •
1kb
R Tc
R Cm
Fig. 1. Restriclion maps of plasmids encoding the cya lcx;us ol B. pertussis. All plasmids are derivatives of pACYCI 84 (Chang and Cohen. 1978). Restriction sites are ordered following cya transcription orientation, Plasmids pDIA4.5,6 permit complementation of a eya strain of E. coli harbouring a plasmid. pVUC-1 (RobertsefaA, 1985) directing synthesis of calmodulin. In acalmodulin-free background, complementation is no longer visible. Plasmids pDIA7 and 8 could not be transformed inio E. coli synthesizing calmodulin, but they directed synthesis of 9. pertussis adenylate cyclase in a calmodulin-free background. Open lines represent the pACYCI 84 vector, solid lines the cya region, and the thin line represents, in pDIA4, an extra Saullla fragment (c. 100bp);inpDIA5the broken line (c. 7 kbp) stands for a B. pertussis DNA fragment that has not been identified as being actually present upstream from (he cya gene.
Bordetella pertussis adenytate cyctase cyclase synthesized from SamHl fragment 4-12, cloned into the unique BamHI site of plasmid pACYC184 (Chang and Cohen, 1978), This fragment was inserted in two orientations (pDIA7 and pDIA8) with respect to tetracycline gene transcription orientation, Adenylate cyclase synthesis differed widely in the two plasmids (see below, Table 1), suggesting that transcription proceeds from site 4 to site 12. Expression of the B. pertussis adenylate cyctase gene from pDIA8 demonstrated that, at least in E. coti. the presence of a positive transcriptional (or translational) effector is not an absolute requirement. Table 1. cAMP synthesis and adenylate cyclase activity ot recombinant clones.
Transforming pla5mid(s)
cAMP nmoles mg"'dry weight bacteria
Adenyjate cyciase aclivity nmoiesmin ' mg ' protein + CaM -CaM
pVUC-IpACYC184 pVUC-1pDIA4 pVUC-1pDIA5 pVUC-1pDiA6 pDIA7 pDIAS
< 0.001 15 43 38 0,01 < 0,001
< 0,01 0,36 0,60 0,25 0,60 0,07
lTyrAiggLuGlyWBl GGGGAACGTTTCAACGTGCGCAAGCAGTTGAACAACGCCAACGTGTATCGCGAAGGCGTG
974
2900 AlaThrGlnThrThcAlaTytGlyLysArgThrGluAsnWBlGlnTycArgHisValGlu GCTACCCAGACAACCGCCTACGGCAAGCGCACGGAGAATGTCCAATACCGCCATOTCGAG LauAlaArgValGlyGlnValValGluValAapThrLeuGluHiBValGlnMABll^llB CTGGCCCGTGTCGGGCAAGTGGTGGAGGTCGACACGCTCGAGCATGTGCAGCACATCATC 3000 , . . . y y y p y GGCGGGGCCGGCAACGA TTCG ATCACCGGCAATGCGCACGACAACTTCCTAGCC GGCOGQ
3100 SBrGlyAapAspAcgLouAspGlyGlyAlBGlyABnABpThrLeuVtlGlyGlyGluCly TCGGGCGACGACAGGCTGGATGGCGGCGCCGGCAACGACACCCTCGTTGGCGGCGAGGGC GlnABnThrVallleGlyGlyAlaGlyABpAspValPhaLeuGlnABpLBuGlyValTrp CAAAACACGGTCATCGGCGGCGCCGGCGACGACGTATTCCTGCAGGACCTGGGGGTATGG 3200 SerAsnGlnLBuAspGlyGlyAlaGlyValAapThcValLyaTyrAanValHiBGlnPro AGCAACCAGCTCGATGGCGGCGCGGGCGTCGATACCGTGAAGTACAACGTGCACCAGCCT SBrGluGluArgLeuGluArgHetGlyAspThcGlyllaHiBAlaABpLeuGlnLyBGly TC CG A GGA GC GCCTC G A AG GC A TGGGC G AC A CGG GC r«TCC A TGC CG A TCTTC A A A A GG GC 3300 . , . . ThrV»lGlui,yarcpPcoAlaL«uA»nL«uPhBS«rValAapHlBWalLy«ABnll»Glu ACGGTCGAGAAGTGGCCGGCCCTGAACCTGTTCAGCGTCGACCATGTCAAGAATATCGAG 3400
994
24
P. Glaser. D. Ladant, O. Sezer, F. Pichot, A. Uilmann and A. Danchin ATCTGC«COGCTCCCGCCT**»CO»CCGCATCGCCGGCaACGACC»GG*C»*CG*CCTC
LcuKBpGlyGIyAapGl.yArgABpThrValAspPna5arClyProGlyArgGlyl.auAap CTCGACGGCGGCGACGGCCGCGATACCGTGGATTTCAGCGGCCCGGGCCGGGGCCTCGAC
p y p y p g v TCOGGCCACGATGCCAACGACACGATACGCGGCCGGGaCCGCGACGACATCCTGCGCGGC
AlaGlyAlaUysGlyValPhELsuSarL-euGlyLysGlyPhaAlaSflrLauHalAapGlu GCCGGCGCAHAGGGCGTHTTCCTGAGCTTGGGCAAGGGGTTCGCCAGCCTGHTGGACGAA
3500
spVal1..*u GTGCTGATCGGCGACGCAGGCSCCAACGTCCTCAATGGCCTGGCGGGCAACGACGTGCTG
noo
3900
.
4600
GluTnrValSarAspAspllcAspGlyGlyAldGlyLauAapThrValAspTyrSerAla GAGACCGTCAGCGATGHCATCGUCGGCGGCGCGGQGCTCGHCACCGTCGACTACTCCOCC JfiOO . . . . MctllsHlBpToGLyAtglleValAlaPraHisGIuTyrGlyPheGlylieGluAlaHsp ATG*TCCATCCAGGCAGGATCGTTGCGCCGC»TGAATACGGCTTCGGG*TCGAGGCGGAC
D
.
ProGluTtirSBriiBtiValLauAcQAanllaGluAanAlaValGlySarAlaAigABpAap CCCGAAACCAGCAACGTGTTGCGCA*TATCG»GAACGCCGTGGGCAGCGCOCGTG*TGAC
TGCAGGACG*C
GGCCTGGGCCTCOJICACCCTGT*TGGCGAGG»CGGCA
1434 1454
IlallaHii! UaHlaAanGlnAlaVatAapGlnAlaGlyllaGluLya'LauifalCluAl* ATCATCCAISCCGCCAACCAGGCGGTAGACCAGGCAGGCATCGAAAA GCTGGTCGA GQC * . . SOOO HHitAlaGlnTyrProAspPtoGlyAlaAlaAlaMaAlaPcoPtoAlaAlaAcgValPro *TGGCGCAGTATCCQGACCCCGGCGCGGCGGCGGCTGCCCCGCCOGCCOCGCeCGTOCCC
167*
AapThrUauHeCGlnSecLsuAlaValAanTcpArg'** GACACGCTGATGCAG TCCCTGGCTGTC AACTGGCGCTGAAGCaCCGTGAATCACGGCCCG 5100 . . . . CCTGCCTCGCGCGGCGGCGCCGTCTCTTTGC GTTCTTCTCC G AG GT » TTTCCC ATC A TG A 5200 CGTCGCCC GTGGC GCAATGCGCCAGCGTGCCCGATTCCGGGTTGCTCTGCCTGGTCATGC
170fi
1B94
TGGCTCGCTATCACGGATTGGCAGCCGATCCCGAGCAGTTGCGGCATGdGTTCGCCGAGC 5100 A G GC A TTCTGT AGCGAAdCGATACAGCCTGGCGGCGCGCCGGGTCGCCCTGAAAGTOCGG CGGCACCGACCCGCGCCGGCGCGGCTGCCACCCGCGCCGCTGCCGGCCATCCCOCTGOdC S400 . . . . CGGCAGQGC GCCTACTTTGTT
i.A^A
Fig. 4. Nucleotide and deduced amino acid sequence of Ihe cya locus ot B. pertussis. The nucleotide sequence represents Ihe coding strand. Numbering of nudeolides starts at ttie A residue of Ihe ATG codon proposed as Ihe translation start of the protein. Two overlapping promoters matching Ihe E. coli consensus sequences (-35: TTGACA, - 10; TATAAT. and transcription start CAT) are indicated by boxes and brackets. The translation slart site has been determined according to criteria defined in the text: the doubly underlined nucleotides are complementary to the3'0H end of £.co/il6S RNA and might act as HBS Two protein domains, Band D. corresponding, as described in Fig. 5. to highly repetitive amino acid sequences are boxed. These sequence data have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00545.
pertussis toxin (Locht and Keith, 1986). A noteworthy feature of the codon usage, which was already prominent for tbe pertussis toxin genes, is the lack of symmetry: for instance, TCG or TAT are frequent codons, whereas their complements, CGA or ATA, are rare. In addition, there is a strong bias against codons ending with A or T. This is very different from the E. coli codon usage. It should be noted, however, that as in £. coli, CTG is the most frequent codon for leucine. These features could be used for assigning ORFs to unknown DNA sequences of B. pertussis: this would be very helpful in view of the fact that a high GC content excludes the presence of frequent translation termination codons. The overall amino acid content of the protein coded by the ORF (M, 177312) does not display any striking features, apart from the absence of cysteine residues and the high content of alanine, glycine, aspartate and asparagine. The absence of cysteine explains the observed resistance of the toxin to W-ethylmaleimide (Friedman ef ai.. 1987). There are no long stretches of hydrophobic amino acids, and the overall amino acid composition of
the protein is not hydrophobic, except perhaps for a signalsequence-like structure 400 amino acids downstream from the N-terminus (see below). Comparison of the protein sequence with itself revealed the existence of four regions or domains (Fig. 5). A first domain, present in pDIA4, of c. 400 amino acids, would correspond to the calmodulin-sensitive catalytic centre. This is followed by a domain of roughly 300 amino acids, very rich in alanine and glycine, and possessing an internal repetition of 13 amino acids (Fig. 5). A sequence of 250 amino acids follows, which has no obvious characteristic features, and the last 700 amino acids constitute a highly repetitive structure, made up of five repetitions of a structure which itself contains an internal glycine/aspartate-rich region repeated 7 to 9 times (see Fig, 5). A 13-amino-acidlong stretch present at the end of the third repetition of glycine repeats is also dearly reminiscent of the 13-aminoacid repeat present in domain B (Fig. 5, middle panel). These sequences have been used to screen protein data banks, and the only clear homology which was apparent was glycine-rich stretches present in several keratin
Bordetella pertussis adenyiate cyclase Table 2. Comparison ot codon usage between the 8. pertussis adenylate cyclase gene (upper figures) and the five pertussis toxin subunit genes (lower figures). F TTT F TTC L TTA L TTG
2 3 40 25 0 1 21
S TCT
CTT
L CTC L CTA
8 7 18 24 3
TAT
Y
TAC
•
TAA
•
TAG
0 1
2 3 10 17 3 3 22 2B
H
CAT
18 11 16 11 14 9 56 24
3 10 40 35 5 5
N AAT
1
S
TCC
s
TCA
s
TCG
23 23 1 2 32 9
It L
Y
3
p
CCT
p
CCC
p
CCA
1
H CAC
a
CAA
Q CAG
16 28 25 35 0 0
C TGT C TGC •
TGA
w
TGG
R CGT
9 6
R CGC
59 33 2 1 22 13
n
CGA
R CGG
L
CTG
96 48
p
CCG
1
ATT
11 9
T
ACT
1
ATC
57
T
ACC
ATA
31 1 7
T
ACA
M ATG
22
T
ACG
31 27
K AAG
32 24
R AGG
A
GCT
D GAT
GCC
A
GCA
87 33
E GAG
56 9 104 29 34 29 50 15
G GGT
A
13 59 112 52 14 12
1
4
V GTT V GTC V GTA V GTG
5 5 45 33 8 7 77 17
A GCG
N AAC K AAA
D GAC E GAA
19 B 49 20 11 4
0 0 0 25 1 4 15 10
s
AGT
2 1
s
AGC
37 36 0 4 4 7
R AGA
G GGC G GGA G GGG
16 5 176 59 12 12 35
25
a signal permitting secretion of the cyclase. U should be noted that several secretion signals occur internally, such as the ovalbumin signal present after the first third of the protein (Lingappa ef ai., 1979) or the opsin signal sequences that are intemal to the protein (Friedlander and Blobel, 1985). No homology could be found either with the other known bacterial cyclase (Aiba ef ai., 1984) or with yeast cyclase (Kataoka ef ai., 1985; Masson ef ai., 1986). The only feature in common with the E coii equivalent protein (848 amino acids) is the presence of a catalytic domain of c. 400 amino acids at the NHs-terminus of the protein (Roy ef al., 1983); this contrasts with the S. eerevisiae enzyme (2026 amino acids), in which the catalytic domain of c. 400 amino acids is carboxy-terminal (Kataoka et ai, 1985; Masson ef ai., 1986). However, as in the yeast enzyme, a highly repetitive sequence was observed, although amino-terminal in yeast and from a completely different motif. This was somewhat surprising and might suggest several pathways for convergent evolution in cAMP synthesis. Homologies with other proteins have been looked for in polypeptide banks (PSeqIP and NBRF) and some homology has been discerned between the first 450 amino acids and nucleotide binding proteins such as dehydrogenases. For instance, a significant homology of 30%, if one takes into account amino acids from the same class (Schwartz and Dayhoff, 1978) was found with E. coii IMP dehydrogenase.
4
The cya gene comprises 1707 codons (including tbe terminal TGA cotJon) whereas tbe sum of pertussis toxin subunits comprises 977 codons Despite the significant difference in amino acid compositian of tbe proleins, it is clear thai the codon usage is very similar in both sets of genes.
genes. This might suggest a filamentous {HoffrTian et ai.. 1985), or periodical (Green and Warren, 1985) structure for the cyclase precursor. Although calmodulin sites are notoriously difficult to identify, the sequence DLLWKIARAGARSAVGTEK displays slight homology with sequence KRRWKKNFIAVSAANRFKKI of skeletal muscle myosin light-chain kinase or RRKWQKTGHAVRAIGRLSSS of smooth muscle myosin light-chain kinase (O'Neil et al.. 1987) and we propose that it corresponds to a peptide interacting with calmodulin. The repetitions present in the domain located downstream from the catalytic domaifi are clustered in two places, bordering polypeptides of 620 and 690 amino acids. This matches the size of the two protein bands present when the B. pertussis cya gene is expressed in E. coii (see Fig. 3). In addition, the corresponding consensus sequence, ASAAAGAVAAA-G, is very similar to the core of signal peptides of proteins exported by £. coli (Gascuel and Danchin, 1986). This might correspond to
Discussion S. pertussis adenylate cyclase is of interest for several reasons: it is a toxin and therefore released extracellularly; it has the highest specific activity reported for any adenylate cyciase (1600 j^mol of cAMP min ^ mg \ Ladant ef ai., 1986); and it is activated by calmodulin. These unusual features raise questions as to the regulation of the activity and evolution of this enzyme. The molecular analysis of the corresponding gene was. therefore, the first step towards answering some of these questions Controversial data on the molecular weight of the enzyme may be relevant to its processing and secretion mechanisms. The purified, extracellular enzyme has been reported to have a molecular weight of 70 kD (Hewlett and WolH. 1976) and, more recently, of 43-50 kD (Shattuck e( at., 1985: Ladant ef ai.. 1986). High-molecular-weight forms of the enzyme were isolated from culture supernatants (Kessin and Franke, 1986). The molecular weight of unaggregated invasive' forms of B. pertussis adenylate cyclase was around 190 kD (Hanski and Fartel. 1985). In addition, we showed that antibodies raised against the 43-50 kD form of the enzyme react in Western blots of virulent S. pertussis extracts with several polypeptides whose molecular weights range between 45 and 200 kD
26
P. Glaser, D. Ladant, 0. Sezer, F. Pictiot, A. Ullmann and A. Danchin
GLGAAPGVPSGRS ASPGLRRPSLGAV VSDMAAVEAAELE A^L QGAQAVAAAQR AASLSAAVFGLGE SSAVAeTVSGFFR 5SRMAGGFGVAGG AMALGGGIAAAVG PDAPAGQKAAAGA ASSIAL.ALAAARG AGAAAGALAAALS SSAYGYEGDALLA KTAAEGAVAGVSA TVGAAVSIAAAAS TGAHAGIAAGRIG
D
ASAAAGAVAAA-G
913 GGDGpDVVLANASRIHYD p 1015 102 4 103 3 104 2 1051 1060 1080 1138 1147 1155 1165 1174 1183 120 3 1262 1271 1280 1289 1298 130 7 1316 1325 134 5 1403 I'll2 1421 14 30 14 39 1448 1457 1477 1529 15 38 154 7 1556 1565 15 74 1583 1604
GGA^TNTVSYAALGRQDSITVSADGERFNVRKQLNNANVYREGVATQTTAYGKRTENVQ GGAGNDSIT GGSGDDRL.D GGAGNDTLV GGEGQNTVI GGAGDDVFLQDLGVWSNQLD GGAGVpTyKYNVHQPSEERLERMGDTGIHADLQKGTVEKHEPALNLFSVDHVKNIENLH GSRLNDRI^A GDDQDNELW \ GHDGNDTIR GRGGGDDILR GGLGLDTLY GEDGNDIFLQDDETVSDDID GGAGLDTVDYSAHIHPGRIVAPHEYGFGIEADLSREWVRKASALGVDYYDNVRNVENVI GTSMKDVLI GDAQANTLH GQGGDDTVR GGDGDDLLF GGDGNDMLY GDAGNDTLY GGLGDDTLE GGAGNDWFGQTQAREHDVLR GGDGVDTVDYSQTGAHAGIAAGRIG_LGILADLGAGRVDKLGEAGSSAYDTVSGIENVV GTELADRIT GD^QA^VLR GAGGADVLA GGEGDDVLL GGDGDDQLS GDAGRDRLY GEAGDDWFFQDAANAGNLLD GGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLHDEPET5NVLRNIENAV GSARDDVLI GDAGANVLN GLAGNDVLS GGAGDDVLL GDEGSDLLS GDAGNDDLF GGQGDDTYLFGVGYGHDTIYE SGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIH
(GGAGDDTL-) '5-7 GGAGDD-FLQD-A-~SD-LD GGAGDVTVDYS(-) , . , G - G I - A D L ( - ) ^ K ( - ) ^ D - V - N - E N V -
consensus
Consensus
Fig, 5. Comparison ol the adenylale cyclase polypeptide with itself. The amino acid sequence ol adenylate cyclase, deduced from the nucleotide sequence (one tetter coda), was compared wilh itself. Stretches ol 15 amino acids having 5 amino acids in common were sought Matching stretches are represented by a diagonal line. Four main domains (upper panel) are revealed by the presence of internal repeats. Domain A (c, 400 amino acids) corresponds !o the catalytic centre of the protein. Domain B (c. 300 amino acids), which can be split in two parts limited by alanlne-rich repeats (middle panel), links the catalytic centre to domain C, which does not display significant repetition. Finally, the lasl 700 amino acids constitute a highly periodical structure (D) consisting of five repeats each containing 7 to 9 glycine-rrch repeats (lower panel). Consensus sequences are determined using identical amino acids (bold letters), and amino acids from Ihe same class (underlined letters), according to Schwartz and Dayhofl (1978),
Bordelella pertussis adenylate cyclase (unpublished results). Our sequence data showed that the gene encodes a polypeptide of 1706 amino acids. Thus, the unprocessed and the non-secreted protein has a much higher molecular weight than the purified enzyme. Our immunochemical results showed that adenylate cyclase expressed in £. coti exhibits a significantly higher molecular weight (between 80 and 120 kD) than the purified extracellular enzyme (Fig, 3). It seems, therefore, that the high-molecular-weight gene product of the S, pertussis cya gene is degraded at its carboxy-terminal end to give a form that retains its ability to be activated by calmodulin. Calmodulin activation is a unique feature of the adenylate cyclase toxins. It is also one of their most intriguing aspects. We have demonstrated that we could reconstitute in vivo the active form of 8, pertussis calmodulin-dependent adenylate cyclase. This was done in a heterologous system involving three partners (a eukaryotic protein, calmodulin, a prokaryotic protein, the B. pertussis adenylate cyclase, and E coti) that allowed the simultaneous synthesis of the two proteins. This was of particular interest since more straightforward reconstitution experiments involving partners of homologous origin, such as S, cerevisiae adenylate cyclase and its presumed RAS protein activator, have led. for instance, to conflicting observations. Uno ef at. (1985) claimed that they could reproduce in E coti the activation of the adenylate cyclase, whereas Kataoka ef at. (1985) found that they failed to reproduce the same activation. Calcium is not an absolute requirement for activation of S, pertussis adenylate cyclase (Greenlee et al., 1982). This contrasts with other mammalian calmodulin-sensitive enzymes, including adenylate cyclases such as the rat brain enzyme. This less stringent interaction may have some relevance to the evolution of calmodulin binding domains. From studies on calmodulin binding sites of mammalian calmodulin-dependent protein kinases, no consensus sequence has been defined (Bennett and Kennedy, 1987). Nevertheless, the existence of an immunological relationship between rat brain and S. pertussis adenylate cyclases argues in favour of a common evolutionary origin of these calmodulin-sensitive enzymes (Monneron et at., unpublished data). We have determined the nucleotide sequence of a single clone carrying the cya locus of B. pertussis. The determined sequence displays one large open reading frame with the potential to encode a very large protein (1706 or 1740 codons, according to the chosen translation start site). Analysis of the predicted amino acid sequence of the protein revealed several striking features. First of all. there do not seem to be the long hydrophobic stretches expected for a protein that is secreted and whose catalytic domain seems to have strong affinity for membranes (Ladant ef al., 1986). Other microbiai adenylate cyclases are primarily membrane-associated (e.g. the E ooli or the
27
S. cerevisiae adenylate cyclases) or cytoplasmic (e.g. the enzymes from Brevibacterium ilquefaciens or Staphytccccous salvarius] (Ide. 1971)). As in the case of E coil and S. oerevisiae adenylate cyclases (Aiba et at., 1964; Kataoka ef al., 1985), membrane localization of B. pertussis adenylate cyclase does not seem to be related to the amino acid composition of the protein, but, rather to some unknown structural feature of the secreted polypeptide. It is worth noting that no signal-peptide-like sequence could be translated from the 5' end of the ORF, such as would be expected for a secreted protein Several sequences with homology to a consensus signal-peptide sequence that might be Involved in secretion, are present downstream from the amino-proximal 400 amino acids. Finally, we find that a carboxy-terminal domain of 700 amino acids consists of repetitions of a glycine-rich motif. This might give periodical or filamentous properties to the protein, and reveal a hydrophobic character, permitting, for instance, exposition of the catalytic centre on the external surface of the cell. A processing system might result in toxin secretion in B. pertussis. This system did not appear to be present in E coli. One is tempted to speculate on another function of the large fragment which would then remain present on the bactenal surface: thus it might have relevance to the haemolytic property of virulent B. pertussis. In this respect, it may be significant that, among Tn5-induced mutations affecting virulence in B. pertussis, Weiss ef al. (1984) have not obtained mutants lacking only adenylate cyclase. These authors obtained either haemotysin-deficient mutants or haemolysin-adenylatecyclase double mutants. If the 5.2 kb ORF encoded a multifunctional protein, the processing, activation and secretion might involve more complex interactions than previously thought. The mechanism for invasion of animal cells by B. pertussis adenylate cyclase has not been elucidated. It is likely that it contributes to the evasion of host defences by suppressing phagocytic functions (Confer and Eaton. 1982). The expression of the adenylate cyclase gene in E coli wiil allow the construction of different truncated genes to check whether the entire gene product or only part of it would enter animal cells. It is possible that adenylate cyclase, like other protein toxins, exhibits a catalytic activity and a function that enables the toxin to interact with the host cells (Middlebrook and Dorland, 1984). The high-molecular-weight gene product (177 kD) in compahson with the 43-50 kD active enzyme, would support such a hypothesis. The successful expression of S, pertussis adenylate cyciase in E coli suggests that it may be possible to apply our novel cloning strategy to a variety of other systems that play a key role in cellular regulation, particularly in caimodulin-sensitive regulatory enzymes, such as protein kinases, phosphodiesterases and adenylate cyclases.
28
P. Glaser. D. Ladant. O. Sezer. F. Pichot, A. Ullmann and A. Danchin
Experimental procedures Bacterial strains and growth media A cya derivative of the restriction-minus E. coti strain, C600SF8 (Struhl ef at., 1976). TP610 was used throughout this work (Hedegaard and Danchin, 1985), Bacteria were grown in rich medium, LB. or minimal medium, M63 (Miller, 1972). Screening for the ability to ferment sugars was performed on MacConkey agar plates containing 1 % of the sugar tested. Antibiotic concentrations were: tetracycline, 10 p.g m l " ' ; chloramphenicoi. 30 ^.g ml"'; ampicillin. 50 jig m\~\ Strain TP610 was transformed (Maniatis ef al., 1982) with plasmid pVUC-1 {a gift from Drs J. Haiech and D,M, Watterson) and checked for calmodulin production. This transformed strain was used as a recipient for cloning a pertussis DNA fragments (strain 18323 W.H.O,).
Screening procedures B. coti strain TP610pVUC-1 is resistant to ampicillin, and the plasmid is from the IncQ incompatibility group. Thus, we chose to clone 6. pertussis DNA in a compatible replicon, pACYC184 (Chang and Cohen, 1978), selecting for chloramphenicoi resistance. Sat/ilia partial digests of total B. pertussis DNA were ligated into pACYC184 DNA cut with SarnHL Clones were screened for maltose fermentation on MacConkey plates supplemented with 100 (i.g ml"' ampicillin, 20 fig m r ' chloramphenicoi. and 1 % maltose. Clones unable to synthesize cAMP cannot ferment maltose, and are white on such plates, whereas clones which produce cAMP ferment maltose and are deep red in colour. Red clones were picked up. purified on the same medium, and checked for p-galactosidase synthesis on LB plates supplemented with the chromogenic p-galactosidase substrate, Xgat. and the lactose operon inducer, IPTG {M'\\\er. 1972); cAMP-deficient bacteria are unable to express the lactose operon. The plasmids were analysed by restriction enzyme digestion and further transformed, either into TP610, where they remained cya-negative, or back into TP610pVUC-1. where cAMP synthesis was restored. When preliminary restriction maps had been obtained, other 6, pertussis DNA fragments were cloned directly into pACYC184. after total restriction with either SamHl or EcoRI, The selection used in the case of EccRI restriction was tetracycljne (10 \xg ml'') because the unique EcoRI site of pACYC184 interrupts the chloramphenicoi resistance gene. For unknown reasons, SamHl cloning always simultaneously yielded several fragments, including the 8,7 kb fragment 4-12 that encompassed the total cya gene. It was possible, however, to clone this fragment alone, albeit in strain TP610 devoid of plasmid pVUC-1. The reason for the calmodulin-induced toxictty is not yet understood.
DNA sequence analysis DNA sequence analysis was performed using subclones in tha phage M13tg131 (Kieny ef ai.. 1983), Unidirectional deletions were generated using the Cyclone System (IBI), The DNA sequence was determined by the dideoxynucleotide method of Sanger e( at. (1977), using {alphaS^^)dATP (Amersham) and 7deaza-dGTP (Boehringer) instead of dGTP, since this results in better resolution of GC-rich regions in polyacrylamide gels (Mizusawa ef at.. 1986), Sequence analysis was performed using
the facilities of the Unitd d'Informatique Scientifiqua of the Pasteur Institute-
Determination of cAMP concentrations Bacteria from an overnight cultura grown In LB medium in the presence of antibiotics were diluted in M63 and heated for 5 min at 100X, Total cAMP was assayed using a standard radioimmmunoassay (Joseph ef at., 1982).
Assay of adenytate cyciase Bacteria from an overnight culture grown in LB medium in the presence of antibiotics were centrifuged, washed with M63 medium, and sonically disrupted in Tris-HCl 20 mM, MgCI? 10 mM, pH 8, After centrifugation, adenylate cyciase was assayed in the supernatants, as described by Ladant ef a/. (1986). The reaction was performed at 30X in a volume of 100 M.1 containing 50 mM Tris-HCl (pH 8) 2 mM (alpha'^P}ATP (5x10^ cpm/assay) 6 mM MgCl2. 100 ii.g bovine serum albumin. 0,13 mM (^H)cAMP (1.5x10" cpm/assay). 0,12 mM CaClj and 0,1 M-M calmoduiin (when added). One unit cf adenylate cyclase corresponds to 1 nmole of cAMP formed in 1 min at 30°C.
Purification and immunologioal detection of adenylate cyclase B. pertussis adenytafe cyclasa was purified from culture supernatants. as described by Ladant ef at. (1986)- Cloned S. pertussis adenylate cyclase was partially purified from E coti strain TP610pDIA7 as follows. An overnight culture in LB medium supplemented with chloramphenicol was centrifuged. and the bacteria were sonically disrupted in Tris HCl 30 mM pH 8, NaCI 15 mM and CaCl2 0,5 mM, 20 ml of the bacterial extract (140 units ml"' adenylate cyciase, 6 mg ml ' protein) were mixed with calmodulin-agarose (Sigma) packed gel and shaken at 4 X for 18 h. The gel which retained 50% of the enzymatic activity was centrifuged and washed with Tris HCl (60 mM) pH 8, NaCI (15 mM), CaClj (0.1 mM) and NP40 (0-1 %), Adenylate cyclase was eluted with the same buffer containing 8 M urea. The eluted adenyiate cyciase was diaiysed overnight against Tris HCl (50 mM) pH 8, CaCl2 (0,1 mM), NP40 (0,1%), For immunoblots. proteins separated by SDS-PAGE (10%) were transferred to nitrocellulose sheets and incubated with anti-8. pertuss/s adenylate cyclase polyctonal serum (dilution 1 /1000) obtained frcm guinea pigs. The detection was performed with '"i-iabelled protein A,
Notes added in proof We have found that the 1300 COOH distai amino acids of the protein are highly hcmoiogous to the tilyA gene product (haemoiysin) of E. coti (Felmiee ef at.. 1985; J Bad 163: 94-105). This suggests fhat both adenylate cyclase and haemoiytic activity are carried by the same protein in B, pertussis.
Acknowledgements We thank J, Haiech and D, M, Watterson for the gift of plasmid pVUC-1. O, Barzu for discussions on the nature of the protein, K, M, Kean for critical reading of the manuscript and for linguistic
Bordetella pertussis adenytate cyclase editing, and G. Limeul for expert typing, Financiai support came from the Centre Nationai de la Recherche Scientifique (UA 1129) and the institut Pasteur (3840 and 3845),
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