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D Cavard

Publications and source records attributed to D Cavard.

At least 37 records · Page 2Linked to original sources
PubMed1985

pH-dependent membrane fusion is promoted by various colicins.

The ability of colicin A, a bacteriocin produced by some Enterobacteriaceae, to fuse phospholipid vesicles at acidic pH, was demonstrated by electron microscopy and resonance energy transfer. The fusion depends on protein concentration and on the nature of the phospholipids. Vesicles, prepared from Escherichia coli phospholipids, fused one or more rounds at pH 4.5 upon addition of stoichiometric amounts of colicin A. Fusion was not only induced by pore-forming colicins (E1, K) but also by colicins that contain nuclease activities (E2, E3). By recombinant DNA technology it is shown that the first glycine-rich 70 NH2-terminal amino acids and, most probably, the extreme COOH-terminal end of colicin A are involved in the fusion activity of the protein. The physiological relevance of this property of colicins is discussed.

Colicins↗
PubMed1985

Lysis protein encoded by plasmid ColA-CA31. Gene sequence and export.

A gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal "signal" region and the mature form is released into the medium together with colicin A.

Amino Acid Sequence↗
PubMed1984

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion.

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of beta-lactamase through genetic recombination. This region comprising amino acid residues 70-335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of beta-lactamase.

Binding Sites↗
PubMed1984

Colicins are not transiently accumulated in the periplasmic space before release from colicinogenic cells.

Colicins are released into the spent medium from colicinogenic cells. The pathway of release has been investigated in this study. The localization in producing cells of colicins A, E3 and of cloacin DF13 has been determined at various times after mitomycin C addition: no transient accumulation in the periplasmic space of colicinogenic E. coli K12 strains was detected by electron microscopy for any of the bacteriocins tested. Furthermore, asynchronous induction in individual cells was detected for each bacteriocin tested. These results strongly suggest that colicins, as well as cloacin DF13, do not transit through the periplasmic space before release from colicinogenic cells.

Bacteriocin Plasmids↗
PubMed1983

Localization of genes responsible for replication and immunity to colicin A on plasmid ColA-CA31.

The region containing the origin and regulatory sites for replication as well as the immunity gene (iaa) have been localized on the plasmid ColA-CA31. The region involved in replication functions of ColA can be hybridized with that of ColE1. It is located between 1 and 1 kb on the plasmid map previously published (Morlon et al. 1982a). A 0.50 Kb HincII fragment of ColA can be weakly hybridized to the ColE1 immunity region. This fragment contains iaa since directed in vitro mutagenesis at an internal restriction site can abolish the immunity to colicin A; however, it does not contain the entire iaa. Knowing the localization of regions involved in autonomous DNA replication and immunity, a mini-ColA plasmid was constructed that contains these two regions. The mini-ColA of 2.8 Kb can be amplified in the presence of chloramphenicol and confers the immunity to transformants. It thus constitutes a useful cloning vector. Expression of ColA and of the various constructed plasmids in the maxicell system suggests that the immunity protein has a molecular weight of about 18-20 Kd.

Cloning, Molecular↗
PubMed1982

[A protease of the external membrane of Escherichia coli sensitive to environmental conditions. Its relations with the expression of envZ gene].

A mutant strain (called Cpr), devoid of the outer membrane protease that cleaves colicin A, has been isolated. The location on the genetic map of the cpr locus as well as its pleiotropic effects concerning chiefly: the protein composition of the outer membrane, sensitivity to phages and colicins, alteration in protease activity (cpr), are very similar to the location on the genetic map and to phenotypic properties observed in strains tpo, perA or envZ which are altered in the ompB locus. Conditions resulting in inhibition of the colicin A protease activity also result in transcriptional regulation of OmpF, OmpC and LamB proteins synthesis. The possibility for this protease to be an osmosensor of the cell's external environment is discussed.

Cell Membrane↗
PubMed1982

A protease as a possible sensor of environmental conditions in E. coli outer membrane.

A mutant strain devoid of the colicin A protease activity (cpr) located at the external face of the outer membrane has been isolated. The location of the mutation (about 74 min on the genetic map), its pleiotropic effects concerning mainly the protein composition of the outer membrane and sensitivity to phages and colicins, are very similar in cpr and in tpo, envZ, perA strains that are affected in the ompB locus. Conditions resulting in inhibition of the colicin A protease activity also result in transcriptional regulation of OmpF, OmpC, and LamB protein synthesis. The possibility for this protease as an osmosensor of the cell's external environment is discussed.

Cell Membrane↗
PubMed1982

Colicins: a minireview.

Colicin are toxins that kill specifically E. coli bacteria. These cells have both an outer and a cytoplasmic membrane. Thus, three steps can be distinguished in colicin action: a) the interaction with specific receptors located at the cell surface, b) the uptake through the outer and eventually through the inner membrane, c) the action of the cell target that leads to cell death. In this short review, an overlook of these three steps is given.

Colicins↗
PubMed1982

Physical map of pColA-CA31, an amplifiable plasmid, and location of colicin A structural gene.

Evidence showing that the plasmic ColA, derived from strain CA31[pColA] can be amplified in the presence of chloramphenicol is presented. This plasmid has been purified and its Mr-value has been found to be 4.6 X 10(6) or 7 kb. Twelve cleavage sites have been mapped in pColA by using single and double restriction endonuclease digestions. These sites were ordered in relation to the single HindIII site. The other restriction endonucleases used were, respectively, SmaI, AvaI, PstI and HincII. Establishment of the map was helped by hybridization of pColA endonuclease digest products with 32P-labeled colicin A-mRNA. The structural gene for colicin A was contained in a 2.17-kb HincII fragment.

Chloramphenicol↗
PubMed1981

Biosynthesis and export of colicin A in Citrobacter freundii CA31.

Synthesis of colicin A after induction with mitomycin C was studied. Specific inhibition of chromosomal protein synthesis occurred very shortly after mitomycin addition. There was no coordinate synthesis of colicin A (61000 Mr) and low-molecular-weight protein. Free and membrane-bound polysome fractions were isolated from cells induced with mitomycin C. Colicin A is synthesized in vitro in the free polysomes and not in the membrane-bound polysomes. Conditions are described which allow a practically specific labelling of colicin A in vivo. By using this system it was possible to demonstrate that colicin A is not transferred cotranslationally across the cytoplasmic membrane. In contrast, this protein leaves the cell where it was made long after synthesis. Preliminary evidence, suggesting that pauses occur during synthesis of colicin A, is presented.

Citrobacter↗
PubMed1981

[Biosynthesis of colicin A : existence of pauses in the translation of messenger RNA].

Evidence has been obtained for the existence of nascent polypeptide chains, that transiently accumulate as discrete size classes, due to discontinuities in the translation process of the colicin A mRNA. Existence of these discontinuities is independent of the hot cell of colicinogenic factor, and from the induction of synthesis by mitomycin C. The information that leads to the existence of discontinuities in the mRNA translation is contained in the plasmid Col A and, very likely, in the Col A gene itself.

Colicins↗
PubMed1981

Exclusive localization of colicin A in cell cytoplasm of producing bacteria.

The production of colicin A in Citrobacter freundii and in Escherichia coli was studied. After induction with low concentrations of mitomycin C, these organisms differed with regards to cell growth, cell viability, and kinetics of colicin A biosynthesis. Despite these differences, immunoferritin labelling on ultra-thin sections of induced frozen cells demonstrated that colicin A was located exclusively within the cell cytoplasm in both types of bacteria. By using protein markers, it was shown that at no time after induction was colicin A accumulated in the periplasmic space or in inner or outer membranes. These results were confirmed by a biochemical approach. For at least 3 h after induction, colicin A remained associated with producing cells and no colicin A activity was found in the periplasmic space. These results are discussed with reference to the synthesis and export of other bacteriocins.

Citrobacter↗
PubMed1979

Purification and molecular properties of a new colicin.

The process of isolation and purification of a new colicin isolated from a Citrobacter strain is described. Escherichia coli sensitive cells are protected by vitamin B12 from the action of this bacteriocin; this suggests that it belongs to the E group of colicins. Therefore, we have called it colicin E4. It has a molecular weight of 56 000 and two molecular forms of isoelectric points 9.4 and 8.2 are separated in electrofocusing on polyacrylamide gels. It has a sedimentation coefficient of 3.4 S and the absorption coefficient A1(280%) nm is 6.23 cm(-1). Using an antibody raised against pure colicin E4, no cross-reaction was detected against colicins A, E1 or K. The physiological effect of colicin E4 on sensitive cells is very similar to that of colicins E1, K or I which disrupt the energized membrane state.

Amino Acids↗
PubMed1977

Role of ionic strength in colicin K adsorption.

The rate of colicin K adsorption to Escherichia coli, and consequent death of the bacteria, is progressively inhibited with increasing ionic strength of the medium. Comparison of the kinetics of colicin adsorption with the kinetics of colicin killing suggests that the lethal event provoked by colicin occurs soon after irreversible colicin adsorption. Factors, such as salts, which protect bacteria against the lethal action of colicin act by preventing colicin adsorption.

Adsorption↗
PubMed1976

Sensitization of colicin K-treated bacteria by sodium dodecyl sulfate: presence of free colicin in colicin K-treated cultures of Escherichia coli.

A method of assaying colicin K is described. It makes use of two properties of sodium dodecyl sulfate to protect bacteria against colicin action and to dissolve those bacteria on which colicin K had started its action. By this method, the kinetics of bacterial killing by colicin K have been measured directly in the treated culture without intervening dilution. The kinetics are exponential with time and are a function of the colicin multiplicity, as described previously, but do not reach a final plateau. At any time during colicin treatment, free colicin is found in the medium. Procedures that eliminate or destroy this free colicin, such as centrifugation and resuspension of the treated bacteria in a fresh medium, or addition of trypsin or sodium dodecyl sulfate to the treated culture stop bacterial killing.

Colicins↗