Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.     

Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin. Three open reading frames were identified downstream from the cyaA gene: cyaB, cyaD and cyaE, coding for polypeptides of 712, 440 and 474 amino acid residues, respectively. As for E. coli alpha-haemolysin, secretion of B.pertussis adenylate cyclase and haemolysin requires the expression of additional genes. The gene products of cyaB and cyaD are highly similar to HlyB and HlyD, known to be necessary for the transport of HlyA across the cell envelope and for its release into the external medium. Complementation and functional studies indicate that the B.pertussis adenylate cyclase-haemolysin bifunctional protein is secreted by a mechanism similar to that described for E.coli alpha-haemolysin, requiring, in addition to the cyaB and cyaD gene products, the presence of a third gene product specified by the cyaE gene.     

Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.     

Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene.

To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells. By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6). Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E. coli. Transformation of plasmid pYC6 into E. coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects. A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome. Two different promoters controlled the production of these three proteins.     

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.     

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

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 B. 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.     

Cloning of the genes of hemolytic factors of Bordetella pertussis in Escherichia coli

The gene library of B. pertussis strain No. 475 (serovar 1. 2. 3.) was obtained on plasmid pUC19 in E. coli strain JM107. The average size of the cloned fragments of chromosomal DNA was 4.9 Kb. Out of 1,700 tested recombinant clones, six caused visible hemolysis on petri dishes with agar containing 4% of sheep red blood cells after 16-hour incubation at 37 degrees C. Only two out of four isolated plasmids were similar in Pst I restriction. Consequently, the existence of several different genes responsible for the hemolytic factors of B. pertussis may be assumed. None of the cloned fragments had Hind III sites. Clones harboring hybrid plasmids possessed different modes of hemolysis, which may be indicative of gene expression in E. coli.     

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.     

Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.     

Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.     

Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase.

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system

The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli

Cloning of higher eukaryotic genes has seldom been performed by complementation of a defective prokaryotic function. This is especially true in the case of functions that are normally absent from the prokaryotic host. We demonstrate here that it is possible to identify by complementation the cDNA from mouse brain, which encodes calmodulin (CaM) synthesis, in spite of the fact that the recipient strain, Escherichia coli, does not normally harbour a CaM function. A three-component cloning procedure in which a gene product requiring CaM for activity, adenylate cyclase from the pathogen Bordetella pertussis, was used to screen a cDNA library for cAMP synthesis in E. coli. The nucleotide sequence of the corresponding cDNA is also reported.     

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX ( r epeats in t o x in) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with non-overlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.     

Maize Glutamine Synthetase Cdnas: Isolation by Direct Genetic Selection in Escherichia Coli

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.     

Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.