Agnes Ullmann

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.

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.

Bordetella pertussis adenylate cyclase: a toxin with multiple talents

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to deliver its amino-terminal catalytic domain into the cytosol of eukaryotic cells. The novelty of the structural organization and conformational flexibility of the CyaA catalytic domain has opened up the way for exploiting this protein as a tool for several biological applications, including epitope delivery, protein targeting and characterization of protein-protein interactions.

One-step purification of hybrid proteins which have β-galactosidase activity

Abstract A one-step purification method of hybrid proteins exhibiting β-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described. Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95%. The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild‐type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse‐labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ‐tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross‐feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co‐ordinate antibiotic biosynthesis.

[5] A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli

Publisher Summary Most biological processes involve specific protein–protein interactions. The yeast two-hybrid system represents a powerful in vivo approach to analyze interactions among macromolecules and screen for polypeptides that bind to a given bait protein. Bacterial equivalents to the yeast two-hybrid system have not been developed yet. This chapter describes a novel bacterial two-hybrid system that allows an easy in vivo screening and selection of functional interactions between two proteins. This system, because of its sensitivity and simplicity, could have broad application in the studies of structure–function relationships in biological macromolecules, in the functional analysis of genomes, and in high-throughput screening of interacting ligands or new therapeutic agents.

On the subunit structure of wild-type versus complemented β-galactosidase of Escherichia coli☆

Abstract Various properties of wild-type galactosidase from Escherichia coli were compared with those exhibited by enzyme extracted from complementary diploids. It is concluded that whereas the complemented enzyme is made by the aggregation of peptide fragments derived from each of the two structural genes present in the diploid, each protomer of the wild-type enzyme behaves as a single covalent unit. The z gene must therefore be regarded as consisting of a single cistron. The mechanism of this apparently new type of intracistronic complementation is briefly discussed.

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.

Délétions fusionnant ľopéron lactose et un opéron purine chez Escherichia coli

From a strain of Escherichia coli K12, which carries an episome F′ and is thereby diploid for the region Lac-Pro2-Ph-T6-Pur, a series of 68 mutants has been isolated in which a fragment of the material carried by the episome is deleted. On one side, all these deletions extend to various sites of gene z. They all cover o, i, Pro2 and Ph. On the other side, 2 of them extend to the region between Ph and T6, 50 extend to the region between T6 and Pur, and 16 extend into one cistron (β), while respecting another cistron (α), of the Pur region. In the 16 deletions which extend into Pur β, the synthesis of the β-galactoside permease and transaeetylase is no longer induced by β-galactosides but repressed by addition of purine in the medium. It appears that in these 16 cases, the deletions have joined a part of the Lac operon to a part of a Pur operon, the new operon which is thus formed being submitted to a repressive regulation by purines.

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 (repeats in toxin) 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.