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C Lazdunski

Publications and source records attributed to C Lazdunski.

At least 91 records · Page 5Linked to original sources
PubMed1986

A molecular genetic approach to the functioning of the immunity protein to colicin A.

A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH2- and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124-143 abolish the protecting function of Cai.

Amino Acid Sequence↗
PubMed1986

Lack of effect of leader peptidase overproduction on the processing in vivo of exported proteins in Escherichia coli.

The kinetics of maturation of certain exported proteins were analysed in Escherichia coli strains that also concomitantly overproduce either a periplasmic protein or the leader peptidase. The results led to three conclusions. Overproduction of leader peptidase has no effect on the rate of maturation of at least two exported proteins, one periplasmic (TEM beta-lactamase), one outer membrane (PhoE); therefore, the quantity of leader peptidase is not rate-limiting for normal export. Overproduction of PhoS reduces the rate of maturation of two other periplasmic proteins (beta-lactamase and PhoA) and itself, presumably by competing for the rate-limiting component of the export apparatus. Overproduction of leader peptidase in a strain overproducing PhoS has no effect on the retarded maturation of PhoS. Therefore even in these conditions, leader peptidase is not rate limiting.

Bacterial Outer Membrane Proteins↗
PubMed1985

Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction.

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.

Chemical Phenomena↗
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

Translation is a non-uniform process. Effect of tRNA availability on the rate of elongation of nascent polypeptide chains.

We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation. In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure. It is linked to the difference in transfer RNA availability for the various codons. Experimental analysis of translation of other proteins in E. coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following. For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster. The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations. The verification of this model and its possible physiological significance are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1984

Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli.

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.

Bacterial Proteins↗
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

The periseptal annulus in Escherichia coli.

Evidence is presented that two circumferential zones of cell envelope differentiation, the periseptal annuli, exist in E. coli as previously observed in S. typhimurium. The periseptal annulus is located at the division site of cells. A strain overproducing a periplasmic protein, PhoS (phosphate-binding protein) has been used to provide a landmark for the periseptal compartment. The zone of adhesion does not involve inner-outer membrane fusion. This zone does not provide a strong physical barrier to protein diffusion in the periplasmic space, at least under conditions of plasmolysis.

Carrier Proteins↗
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

Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate.

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

Amino Acid Sequence↗
PubMed1983

Voltage and time dependence of the conductance of planar lipid bilayers doped with colicin A.

Voltage jump studies of planar bilayers doped with colicin A revealed two classes of time and voltage dependent conductance induced by colicin A. The first type of conductance, asymmetrically voltage dependent, measured as a time dependent process, was in agreement with previously described conductances of bilayers doped with colicin A or colicin E1. The second class of conductance appeared as a voltage dependent instantaneous conductance. The significance of this new type of conductance is discussed.

Colicins↗
PubMed1983

Nucleotide sequence of promoter, operator and amino-terminal region of caa, the structural gene of colicin A.

The nucleotide sequence of 378 bp covering the promoter-operator regions and the region coding for the N-terminal portion of the colicin A gene was determined. These assignments were made possible by the determination of the N-terminal 12 amino acids of the colicin A protein. DNA sequence homologies between operator regions of recA, lexA, uvrA, uvrB, cea and caa genes are discussed.

Amino Acid Sequence↗
PubMed1983

Isolation, molecular and functional properties of the C-terminal domain of colicin A.

Partial proteolytic digestion of colicin A with bromelain allowed the isolation of a 20-kd fragment. This fragment has been purified to homogeneity and its molecular properties have been studied. The sequence of the 54 N-terminal amino acid residues has been determined by automated Edman degradation. This sequence is identical to that of the predicted amino acid sequence of the 20-kd C-terminal part of the colicin A polypeptide deduced from the nucleotide sequence of the caa gene. This polypeptide can produce channels in phospholipid planar bilayers of the same size as those formed by colicin A. However, the voltage-dependence for opening and closing was drastically altered in the peptide fragment channels. The latter, in contrast to colicin A channels, remained open over a wide range of voltage. Large negative potentials were required to close the peptide fragment channels although opening took place in the same voltage range as for colicin A ionic pores.

Amino Acid Sequence↗
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

Membrane potential (delta psi) depolarizing agents inhibit maturation.

Precursor forms of exported proteins were first accumulated in the envelope of phenethyl alcohol (PEA)-treated cells. After removal of PEA, a complete processing could be obtained in a few minutes. In this work, we demonstrate that colicins A and E1, that act on the electrical gradient in the cytoplasmic membrane, prevent the processing of precursor forms previously accumulated. Concentrations of colicins accounting for approximately 1 killing unit (50--3000 molecules/cell) were found to be sufficient for inhibition of processing. Therefore our results strongly suggest that in intact cells the electrical gradient across the cytoplasmic membrane is required for maturation of exported proteins.

Bacterial Proteins↗