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J Bellalou

Publications and source records attributed to J Bellalou.

13 recordsLinked to original sources
PubMed2001

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli.

BACKGROUND: To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. RESULTS: To overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate. CONCLUSIONS: This approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Adaptation, Physiological↗
PubMed1996

Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis.

In Bordetella pertussis, transcription of virulence-associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal-transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility-shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the target for BvgA-P binding, and probably contains multiple binding sites.

Adenylate Cyclase Toxin↗
PubMed1995

Interaction of calcium with Bordetella pertussis adenylate cyclase toxin. Characterization of multiple calcium-binding sites and calcium-induced conformational changes.

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Adenylate Cyclase Toxin↗
PubMed1994

Mutations in the Bordetella pertussis bvgS gene that confer altered expression of the fhaB gene in Escherichia coli.

The bvg locus of Bordetella pertussis, required for coordinate regulation of virulence genes in response to environmental signals, encodes two proteins, BvgS and BvgA, that belong to the bacterial two-component signal transduction systems. We have isolated spontaneous mutations of the bvg locus in Escherichia coli and analyzed their effects on the expression of fhaB::lacZY transcriptional fusions. The mutations, localized in the linker and transmitter domain of BvgS, result in increased activation of fhaB and/or in insensitivity to a modulating agent, nicotinic acid.

Amino Acid Sequence↗
PubMed1992

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.

Adenylate Cyclase Toxin↗
PubMed1990

Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase.

The Bordetella pertussis cyaA gene encodes a virulence factor which is a bifunctional protein exhibiting calmodulin-sensitive adenylate cyclase and hemolytic activities (P. Glaser, H. Sakamoto, J. Bellahov, A. Ullmann, and A. Danchin, EMBO J. 7:3997-4004, 1988). We characterized the hemolytic and toxin activities of the 200-kilodalton (kDa) bifunctional (CyaA) protein and showed that, whether cell associated or secreted, the 200-kDa CyaA protein carries hemolytic and toxin functions. The catalytically active 45-kDa form of adenylate cyclase released by proteolytic digestion of the 200-kDa CyaA protein displayed neither hemolytic nor toxin activities. We constructed in-phase deletions in the 3' region of the cyaA gene, which presumably carries the hemolytic determinant, and showed that the resulting proteins exhibited wild-type adenylate cyclase activity and were secreted without processing into culture supernatants. The hemolytic activities of these mutant CyaA proteins were severely reduced, and their toxin activities were abolished. These results suggest that the structural integrity of the 200-kDa CyaA protein is necessary for toxin activity and that distinct structural determinants within the CyaA protein are involved in secretion, pore formation, and entry into target cells.

Adenylyl Cyclases↗
PubMed1990

Synthesis and secretion of Bordetella pertussis adenylate cyclase as a 200-kilodalton protein.

Bordetella pertussis, the etiological agent of whooping cough, synthesizes a calmodulin-sensitive adenylate cyclase that is suspected to play a major role in the virulence of this bacterium. We show that adenylate cyclase synthesized as a 200-kilodalton protein is the product of the cyaA gene and that various virulent Bordetella species secrete this high-molecular-weight polypeptide without apparent proteolytic processing. When submitted to trypsin digestion, the 200-kilodalton protein was converted to a stable 45- to 50-kilodalton species. This corresponds to the size of the enzyme previously purified from a culture supernatant. The molecular heterogeneity reported for the various identified forms of adenylate cyclase could therefore result in part from proteolytic degradation or molecular aggregation of the major 200-kilodalton form of the enzyme.

Adenylyl Cyclases↗
PubMed1988

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.

Adenylate Cyclase Toxin↗
PubMed1988

Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin.

An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.

Adenylyl Cyclases↗
PubMed1988

Immunological relatedness between Bordetella pertussis and rat brain adenylyl cyclases.

A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin. Making use of polyclonal antibodies raised against the bacterial adenylyl cyclase and the rat brain adenylyl cyclase catalytic component, respectively, we showed an immunological cross-reactivity between the two enzymes. Furthermore, B. pertussis adenylyl cyclase was inhibited and immunoprecipitated by the homologous and one of the heterologous immune sera. These results suggest an evolutionary relationship between the B. pertussis enzyme and its eukaryotic counterpart.

Adenylyl Cyclases↗
PubMedDate not supplied

Studies on culture conditions of Bordetella pertussis and relationship to immunogenicity of vaccines.

Conditions for growing Bordetella pertussis bacteria in fermentors were studied for the purpose of producing highly protective vaccines. Bacteria with high protective potency were obtained in fermentors when the inoculum consisted of a dense suspension of young B. pertussis cells grown on Bordet-Gengou agar plates. When the time of cultivation in fermentors exceeded 30 h, bacteria harvested showed lower protective potency activity and lower levels of histamine sensitizing factor (HSF) and leukocytosis promoting factor (LPF), indicating that the optimal time for collection is at the end of the logarithmic growth phase. In fermentors, the ATP concentration in the bacterial cells increased from the time of inoculation until the end of the logarithmic growth phase. At the late logarithmic growth phase, the amounts of ATP, HSF, LPF and protective antigen declined rapidly. ATP kinetics thus provides a way of predicting the immunogenicity of vaccines. These results may be applicable to the production of vaccines exhibiting high protective activity at low bacterial concentrations, and to the preparation of soluble vaccines resulting from high-yield extraction of protective antigens. A correlation between the potency and LPF activity of vaccines was established for use in the rapid screening of vaccines for protective activity.

Animals↗
PubMedDate not supplied

Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

Bioreactors↗