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At least 73 records · Page 4Linked to original sources
PubMed1985

Studies of colicin action on wall-less stable L-forms of Escherichia coli. III. A colicin-tolerant mutant in a wall-less stable L-form.

The conversion of a tol III Escherichia coli mutant, tolerant to colicins E2, E3, Ia and K, into protoplast-like stable L-form makes it sensitive to colicins E2, Ia and K. Its original sensitivity to colicin E1 and tolerance to E3 are preserved in this cell form. Thus, rods of this mutant are not truly tolerant to colicins E2, Ia and K, but "pseudotolerant": their wall receptors have been turned out to "nonlethal" ones by the mutation in question.

Colicins↗
PubMed1986

Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity.

Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.

Amino Acid Sequence↗
PubMed2003

Colicin S8 export: extracellular and cytoplasmic colicin are different.

The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process.

Chromatography, Ion Exchange↗
PubMed1983

Nucleotide sequence for the catalytic domain of colicin E3 and its immunity protein. Evidence for a third gene overlapping colicin.

We have determined the nucleotide sequence of a segment of Co1E3 DNA coding for the carboxyl-terminal, catalytic peptide of colicin E3 and for the immunity protein. The end of the colicin E3 gene is separated from the beginning of the immunity gene by a nine-basepair intercistronic region, suggesting the two genes are expressed as a single transcriptional unit. The immunity gene is expressed, however, in E. coli strains containing Co1E3-pBR322 hybrid plasmids deleted for the 5'-end of the colicin gene. The DNA sequence also contains an unexpected open reading frame (ORF). This ORF is contained within the colicin gene and is in the +1 reading frame with respect to that gene. Plasmids containing the ORF, directed the synthesis of an 11 kilodalton protein in a cell-free, transcription-translation system.

Amino Acid Sequence↗
PubMed1974

Studies on the regulation of colicin Ib synthesis: replication of the Col Ib-P9 plasmid during colicin induction.

The Col Ib-P9 plasmid content of colicinogenic cells has been measured by both sedimentation analysis of cleared lysates and by deoxyribonucleic acid hybridization to purified Col Ib-P9 factor deoxyribonucleic acid. The results show an independence between mitomycin C-induced colicin synthesis and Col Ib-P9 replication. In addition, the ratio of the various molecular configurations (i.e., covalently closed circles or open circles) of the Col Ib-P9 plasmid does not change upon induction of colicin synthesis. Heat treatment of a colicinogenic strain carrying a thermosensitive dnaB mutation on the bacterial chromosome leads to complete inhibition of Col Ib-P9 plasmid replication and the simultaneous induction of colicin Ib synthesis in all cells of the colicinogenic population. These studies rule out mechanisms or regulation of colicin Ib synthesis based on the induced replication of the Col Ib-P9 plasmid. Our studies show that in a Col Ib-P9 colicinogenic population there is, on the average, one plasmid per cell.

Colicins↗
PubMed1973

Reversibility of the specific adsorption of colicin E2-P9 to cells of colicin-sensitive strains of Escherichia coli.

The adsorption of colicin E2-P9 to its specific receptors on cells of sensitive strains of Escherichia coli is reversible under normal experimental conditions. At temperatures above 20 C, colicin may desorb from one cell and be readsorbed by a second with potentially lethal consequences. However, desorption of colicin seems unable to rescue a cell once it has received a lethal dose. These findings have implications both for the nature and types of specific receptors, and for the assay of colicin by the survivor count (lethal unit) methods.

Adsorption↗
PubMed1978

Colicins and bacterial membranes: structures and functions. I. Effects of colicins on the protein composition of the membranes of sensitive and tolerant Escherichia coli.

Treatment of Escherichia coli K12 C600 with colicin K or E1, but not E3, caused changes in the protein composition of the bacterial cytoplasmic membrane and an impairment of the membrane-associated ATP-linked transhydrogenase activity. The major compositional changes were loss and/or reduction in the levels of protein bands 4, 8, 9, 10, 13, and 18 with approximate molecular weights of 122,000, 81,000, 75,000, 73,000, 62,000, and 44,000, respectively. Colicin K or E1 treatment had no significant effect on the protein composition or the ATP-linked transhydrogenase activity of the cytoplasmic membranes of the isogenic tolerant strain E. coli K12 C600 TolII (A592). The cytoplasmic membranes of the untreated tolerant mutant were characteristically devoid of protein bands 4 and 13. It is proposed that protein bands 4 and/or 13 participate in colicin action by acting as receptors for colicins at the cytoplasmic membrane level. Some observations on the structural and functional heterogeneity of the cytoplasmic membrane preparations were made.

Adenosine Triphosphate↗
PubMed1979

Mode of action of colicin E2, colicin E3 and cloacin DF13.

The bacteriocins colicin E2, colicin E3 and cloacin DF13 are bactericidal proteins which are excreted by producing cells as a complex of two polypeptides, present in equimolar amounts. The high-molecular weight component is responsible for the biochemical lesion induced by these bacteriocins. The low-molecular weight component, the so-called immunity protein, was shown to act as inhibitor of the high-molecular weight component. Colicin E2, freed from immunity protein, causes endonucleolytic degradation of DNA. Colicin E3 and cloacin DF13, freed from immunity protein, induce ribosome inactivation by endoribonucleolytic cleavage of 16S rRNA. A possible role of the immunity protein in the killing activity of the bacteriocins is not quite clear. Probably it keeps the high-molecular weight component in the right configuration for penetration of the cell wall layers. Studies on structure-function relationships have shown that cloacin DF13 has a domain-type structure. Various properties have been located in the hydrophilic C-terminal part of the molecule.

Bacterial Proteins↗
PubMed1991

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.

Bacterial Proteins↗
PubMed1997

The long and short of colicin action: the molecular basis for the biological activity of channel-forming colicins.

Channel-forming colicins undergo a remarkable series of conformational gyrations during their voyage from the extracellular milieu to the periplasmic membrane. Crystal structures of the intact colicin la molecule and a channel-forming domain of colicin E1 illuminate relationships between the molecular structure and biological function of these voltage-dependent channel-forming toxins.

Bacterial Proteins↗
PubMed1985

Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

The channel-forming activity of colicin E1 in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that this pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin E1 and a more slowly binding channel-forming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and less than 4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [3H]leucine-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH less than 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of one or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin E1. This model may also be relevant to the mechanism of membrane insertion by certain toxins.

Colicins↗
PubMed2002

Mutagenic scan of the H-N-H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases.

Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H-N-H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H-N-H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H-N-H motif is the site of DNA binding and that Mg2+-dependent cleavage of double-stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation-dependent catalytic mechanisms. The roles of individual amino acids within the H-N-H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase-activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H-N-H DNases.

Alanine↗
PubMed1984

Nucleotide sequence of the gene for the immunity protein to colicin A. Analysis of codon usage of immunity proteins as compared to colicins.

The nucleotide sequence of the structural gene for the immunity protein to colicin A (cai) has been established. This sequence consists of 534 base pairs. According to the predicted amino acid sequence, the polypeptide chain of this immunity protein comprises 178 amino acids and has a relative molecular mass of 20462. As expected from its localization in the inner membrane, large hydrophobic fragments are found along the polypeptide chain that also contains clusters of mostly positively charged residues. The cai like the ceiA genes encode proteins that are weakly expressed as compared to the corresponding colicins (A and E1). Codon usage reflects this difference. In contrast, the four genes for immunity to cloacin DF13 and to colicin E3 and for these bacteriocins, all of which are highly expressed and are organized in operon, display similar codon usage. These results are discussed with regards to the possible relationship between expressivity and codon usage.

Bacterial Proteins↗
PubMed2006

Prevalence of ColE1-like plasmids and colicin K production among uropathogenic Escherichia coli strains and quantification of inhibitory activity of colicin K.

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.

Bacterial Proteins↗
PubMed1975

Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group B.

This paper describes the isolation of mutants resistant to colicins of group B (i.e., colicins B, D, G, H, Ia, Ib, M, Q, S1, and V). The 145 mutants studied in detail can be divided into nine phentopyic classes, based on their colicin-resistance patterns. They include the previously isolated tonA, tonB exbB and cir mutants. Each of the different phenotypic classes of mutants has been partially characterized, and some approximately mapped. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed substantial changes in the composition of the outer membrane proteins of exbB and tonB mutants.

Anti-Bacterial Agents↗
PubMed1978

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.

Colicins↗