The effect of nonreceptor adsorption on the lethal action of colicin E1

The survivability of Escherichia coli K12s cells has been studied after treatment with 125I-labeled colicin E1. It has been shown that for low amounts of adsorbed colicin the survivability follows single-hit kinetics. When the number of colicin molecules adsorbed exceeds approx. 50 per cell, deviation from single-hit kinetics occurs towards higher survivability. Colicin E1 adsorbed nonreceptorwise by the cell's surface has been shown to inhibit the lethal action of colicin E1 molecules adsorbed at specific receptors. This fact has been used in accounting for the elevated survivability of cells at high colicin doses. The functional significance of the phenomenon is discussed.     

Kinetics of Lethal Adsorption of Colicin E2 by Escherichia coli

The kinetics of lethal adsorption of colicin E2 by Escherichia coli C6 were studied by means of survivor plots. These were determined by a method which allowed rapid sampling of the reaction mixture and estimation of approximate confidence limits for the plotted data. The results were consistent with the predictions of a hypothetical model that assumed a single-hit mechanism of colicin action upon a bacterial population whose cells varied in their number of specific (lethal) receptors for colicin. The possibility of nonlethal adsorption is discussed.     

Fragmentation of colicins A and E1 by cell surface proteases.

Interaction of either colicin A or E1 with the surface of Escherichia coli cells resulted in extensive cleavage of the colicins into many peptide fragments in the molecular weight range of 10,000 to 30,000 released into the supernatants of colicin-cell mixtures. The protease inhibitor P-aminobenzamidine inhibited the cleavage of colicin A and enhanced colicin killing activity, suggesting that proteolysis is not required for the killing action of colicin. Fragments derived from the supernatants of the mixtures were inactive against sensitive cells. Proteolytic activity against both colicins was localized primarily in the outer membrane fraction of the cell envelope. At least two distinct protease activities appear to be present. Examination of the patterns of cleavage and inactivation of the colicins by a series of resistant mutants indicates that specific colicin receptors play no essential role in colicin proteolysis. In addition, evidence is presented that adsorption of colicin to specific receptors is a reversible process.     

Kinetics of Adsorption of Colicin CA42-E2 and Reversal of its Bactericidal Activity

The kinetics of killing of Escherichia coli K-12 by colicin CA42-E2 have been studied, and the data were used to estimate the adsorption constant of this colicin under various environmental conditions. Evidence was obtained suggesting that the adsorption of colicin occurred in two stages; the earlier stage was reversible and did not lead to the death of the cell, the latter stage was irreversible and bactericidal. Cells which had adsorbed a lethal quantity of colicin could be rescued for a short time by inactivating the adsorbed colicin with trypsin. However, when the metabolic activity of the cells was totally arrested the lethal effect of adsorbed colicin was subject to trypsin reversal over long periods of time.     

Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro.

Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 43 degrees C but was only slightly affected by variation of the pH between 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin E1 or colicin E3.     

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.     

The cytotoxic and cytocidal effect of colicin E3 on mammalian tissue cells

The plasma membrane of mammalian cells can mediate the cytotoxic and cytocidal effects of colicin E3. As little as 10² lethal units of purified colicin E3 per cell exert a pronounced cytocidal effect on human epithelial HeLa cells and as little as 10⁴ lethal units per cell also on line L mouse fibroblasts in tissue culture. Cells in complete monolayers are rapidly killed, become spherical and shrink, they are detached from the support and finally autolyzed. The percentage of killed cells in both lines is directly proportional to the multiplicity of colicin used. Theld ₅₀ for HeLa cells is about 30 times lower than for L cells. At the multiplicity of 10⁵ l.u., usually 100 % HeLa cells and 90 % L cells are killed in 2–3 days. Purified colicins E2 and D have no demonstrable cytological effect on HeLa cells, although DNA synthesis in L cells appears to be partly inhibited by colicin E2. The profound effect of colicin E3 on mammalian cells could be interpreted in a similar way as in bacteria,viz. as a specific cleavage of rRNA.     

Studies of colicin action on wall-less stable L-forms of Escherichia coli. I. Degree of attachment and of killing effect on rods and stable L-form cells.

Escherichia coli strains B and K12 W 1655 F+ are able to bind more lethal units of colicins E2, E3, G, H, Ia, and K+ X per one stable L-form cell (of the protoplast type) than per one rod cell; colicin D is bound in a higher amount on E. coli B rods. This pattern remains unchanged, if the same colicins are attached on chloroform-killed cells of both forms. Rods of both E. coli strains are more sensitive to colicins D, E2, E3, K + X (as--in the strain B--to colicin Ia) than cells of the respective L-forms. In the strain W 1655 F+ both cell forms are equally highly sensitive to colicin Ia. The stable L-forms of both strains are much more sensitive to colicins G and H than the rods. Thus the Gram-negative cell wall decreases the probability of a colicin molecule to get attached to its receptor in the cytoplasmic membrane. On the other hand, in E. coli cells the attachment of most colicin molecules to the wall receptors increases the probability of their biological effect. There is no such effect of the wall-attachment on the action of colicins G or H. The strain B is tolerant to colicin E2, while being resistant to E3; thus the cytoplasmic membrane receptor sites for them are not identical.     

Colicin M is only bactericidal when provided from outside the cell

Summary The colicin M structural gene, cma, was subcloned in a vector which allowed temperature-inducible control of its expression. Induction of expression of cma in colicin M uptake proficient strains was lethal for the host cell when the colicin M immunity protein was not present. In liquid culture cells lysed, and no colonies were formed on solid media. These effects were not observed in mutants defective in the colicin receptor (FhuA) or uptake functions (TonB, TolM), nor in wild-type cells treated with trypsin prior to induction of cma expression. It was concluded that cytoplasmic colicin M is not toxic for the producing cell. To exert a lethal effect the colicin has to enter the cell from outside. Cells expressing cma released small amounts of colicin M.     

Interaction of Colicins with Bacterial Cells IV. Immunity Breakdown Studied with Colicins Ia and Ib

Colicinogenic cells are immune to the lethal effect of the colicin which they produce. In the presence of very high concentrations of colicin, however, colicinogenic cells are no longer immune to the homologous colicin. This phenomenon, immunity breakdown, was studied with colicins Ia and Ib. The biochemical effects of colicin Ib on Escherichia coli were studied with a standard noncolicinogenic strain. At multiplicities of about 10 or higher, colicin Ib inhibited incorporation of leucine into protein and incorporation of (32)P-inorganic phosphate into deoxyribonucleic acid and ribonucleic acid by more than 95%. Under the same conditions, (32)P incorporation into phospholipid and nucleotide fractions was inhibited only partially (about 80 and 60%, respectively). Inhibition of (32)P incorporation into the terminal phosphorus of adenosine triphosphate was also considerably less than that of macromolecular synthesis (50 to 60%). (32)P incorporation into the nonnucleotide organic phosphate fraction was not inhibited. Respiration was not affected. Colicin Ia showed the same biochemical effects as colicin Ib. A mutant of an Ib-colicinogenic E. coli strain selected for resistance to low concentrations of colicin Ia was shown to be resistant to high concentrations of homologous colicin Ib, whereas the parent Ib-colicinogenic strain is sensitive to high concentrations of colicin Ib. This mutant lost its specific receptors for colicin Ib. Moreover, the biochemical effects of high concentrations of colicin Ib on Ib-colicinogenic cells during immunity breakdown were similar to the effects found in sensitive cells exposed to low concentrations of the same colicin. It is concluded that the killing of colicinogenic cells in the presence of high concentrations of homologous colicin is indeed caused by the homologous colicin molecules.     

Interaction of 125I-Labeled Colicin E1 with Escherichia coli

Colicin E1 protein was labeled with ¹²⁵I to specific activities of up to 2 × 10⁸ cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.     

Transport of Vitamin B12 in Escherichia coli: Common Receptor Sites for Vitamin B12 and the E Colicins on the Outer Membrane of the Cell Envelope

The first step in the transport of cyanocobalamin (CN-B(12)) by cells of Escherichia coli was shown previously to consist of binding of the B(12) to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B(12) receptor sites also function as the receptors for the E colicins, and that there is competition between B(12) and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B(12) auxotroph, and B(12) transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B(12) to the outer membrane B(12) receptor sites, and CN-B(12) protected cells against these colicins. Half-maximal protection was given by CN-B(12) concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of (57)Co-labeled CN-B(12) to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B(12) receptors. Colicin K did not inhibit the binding of B(12) to the outer membrane receptor sites, and no evidence was found for any identity between the B(12) and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B(12) transport, which is believed to consist of the energy-coupled movement of B(12) across the inner membrane.     

Identification of a unique specificity determinant of the colicin E3 immunity protein.

Plasmid immunity to a nuclease-type colicin is defined by the specific binding of an immunity (or inhibitor) protein, Imm, to the C-terminal nuclease domain, T2A, of the colicin molecule. Whereas most regions of colicin operons exhibit extensive sequence identity, the small plasmid region encoding T2A and Imm is exceptionally varied. Since immunity is essential for the survival of the potentially lethal colicin plasmid (Col), we inferred that T2A and Imm must have co-evolved, retaining their mutual binding specificities. To evaluate this co-evolution model for the col and imm genes of ColE3 and ColE6, we attempted to obtain a stabilized clone from a plasmid which had been destabilized with a non-cognate immunity gene. A hybrid Col, in which the immE3 gene of the ColE3 was replaced with immE6 from ColE6, was lethal to the host cells upon SOS induction. From among this suicidal cell population, we isolated a stabilized, i.e., evolved, clone which produced colicin E3 (E3) stably and exhibited immunity to E3. This change arose from only a single mutation in ImmE6, from Trp48 to Cys, the same residue as in the ImmE3 sequence. In addition, we constructed a series of chimeric genes through homologous recombination between immE3 and immE6. Characterization of these chimeric immunity genes confirmed the above finding that colicins E3 and E6 are mostly distinguished by only Cys48 of the ImmE3 protein.     

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.     

Colicin biology.

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.     

Protection of Escherichia coli from the lethal effect of colicins by high osmotic pressure

The sensitivity of Escherichia coli to the lethal effect of colicin E(2) was reduced by elevation of osmotic pressure of the incubation medium. Optimal protection of the cells from the lethal effect of colicin E(2) was achieved with 0.6 to 0.8 m NaCl or with 0.8 m sucrose containing 0.01 m MgSO(4). Under such conditions, the degradation of deoxyribonucleic acid caused by colicin E(2) was also suppressed markedly. It was concluded that a high concentration of sucrose with Mg(++) might prevent the action of the adsorbed colicin E(2). A similar protection was observed against the lethal effect of colicin K.     

Novel colicin Fy of Yersinia frederiksenii inhibits pathogenic Yersinia strains via YiuR-mediated reception, TonB import, and cell membrane pore formation

A novel colicin type, designated colicin Fy, was found to be encoded and produced by the strain Yersinia frederiksenii Y27601. Colicin Fy was active against both pathogenic and nonpathogenic strains of the genus Yersinia. Plasmid YF27601 (5,574 bp) of Y. frederiksenii Y27601 was completely sequenced. The colicin Fy activity gene (cfyA) and the colicin Fy immunity gene (cfyI) were identified. The deduced amino acid sequence of colicin Fy was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin Fy-susceptible strain Yersinia kristensenii Y276 revealed the yiuR gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin Fy receptor molecule. Introduction of the yiuR gene into the colicin Fy-resistant strain Y. kristensenii Y104 restored its susceptibility to colicin Fy. In contrast, the colicin Fy-resistant strain Escherichia coli TOP10F' acquired susceptibility to colicin Fy only when both the yiuR and tonB genes from Y. kristensenii Y276 were introduced. Similarities between colicins Fy and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin Fy and colicin Ib producers suggest a common evolutionary origin of the colicin Fy-YiuR and colicin Ib-Cir systems.     

Colicin Biology

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.     

Relationship between the transport of iron and the amount of specific colicin Ia membrane receptors in Escherichia coli.

Strains of Escherichia coli K-12 defective in their ability to utilize exogenously supplied iron due to genetic defects in the entF, tonB, fes, or fep gene exhibited elevated levels of the specific outer-membrane receptor for colicin Ia when compared with parental strains. Although entF, fes, and fep strains showed a higher degree of Ia sensitivity than did the parental strains, tonB strains were resistant to colicin action. The colicin insensitivity of tonB strains was not due to hyperproduction of enterochelin. Growth in medium containing 101.8 muM Fe2+ led to a lowering of receptor levels in all the above strains and resulted in decreased colicin Ia sensitivity in all strains except tonB, which was already at maximal resistance. Growth in citrate plus iron (1.8 muM) or in ferrichrome resulted in a substantial reduction in both receptor levels and Ia sensitivity in ent, fes, and fep strains but had no effect on receptor levels in tonB strains. Growth in citrate did not lead to an alteration in receptor levels in a mutant specifically defective in citrate-mediated iron transport. The presence of enterochelin during growth led to a reduction in the number of receptors in the parental and ent strains but not in tonB, fes, or fep strains. Thus, in all cases examined, there was an inverse relationship between the number of colicin receptors per cell and the ability of the strain to take up iron from the growth medium. This suggests that under conditions of iron limitation there is a derepression of colicin Ia receptor biosynthesis. These results may point to a role of the colicin I receptor in iron uptake.     

Crystal structure of colicin E3: implications for cell entry and ribosome inactivation.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.