Colicin E3 is an endonuclease.

It was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation. The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifically inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.     

Colicin M is inactivated during import by its immunity protein

Colicin M (Cma) displays a unique activity that interferes with murein and O-antigen biosynthesis through inhibition of lipid-carrier regeneration. Immunity is conferred by a specific immunity protein (Cmi) that inhibits the action of colicin M in the periplasm. The subcellular location of Cmi was determined by constructing hybrid proteins between Cmi and the TEM--lactamase (BlaM), which confers resistance to ampicillin only when it is translocated across the cytoplasmic membrane with the aid of Cmi. The smallest Cmi'-BlaM hybrid that conferred resistance to 50 g/ml ampicillin contained 19 amino acid residues of Cmi; cells expressing Cmi'-BlaM with only five N-terminal Cmi residues were ampicillin sensitive. These results support a model in which the hydrophobic sequence of Cmi comprising residues 3–23 serves to translocate residues 24–117 of Cmi into the periplasm and anchors Cmi to the cytoplasmic membrane. Residues 8–23 are integrated in the cytoplasmic membrane and are not involved in Cma recognition. This model was further tested by replacing residues 1–23 of Cmi by the hydrophobic amino acid sequence 1–42 of the penicillin binding protein 3 (PBP3). In vivo, PBP3'-'Cmi was as active as Cmi, demonstrating that translocation and anchoring of Cmi is not sequence-specific. Substitution of the 23 N-terminal residues of Cmi by the cleavable signal peptide of BlaM resulted in an active BlaM'-'Cmi hybrid protein. The immunity conferred by BlaM'-'Cmi was high, but not as high as that associated with Cmi and PBP3'-'Cmi, demonstrating that soluble Cmi lacking its membrane anchor is still active, but immobilization in the cytoplasmic membrane, the target site of Cma, increases its efficiency. Cmi1-23 remained in the cytoplasm and conferred no immunity. We propose that the immunity protein inactivates colicin M in the periplasm before Cma can reach its target in the cytoplasmic membrane.     

Use of Colicin E3 for Biological Containment of Microorganisms

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms.     

Incompatibility Exhibited by Colicin Plasmids E1, E2, and E3 in Escherichia coli

Escherichia coli strains were made multiply colicinogenic for the colicin plasmids E1, E2, or E3 (Col E1, Col E2, or Col E3, respectively) by both a deoxyribonucleic acid transformation system and bacterial conjugation. The multiply colicinogenic bacteria constructed exhibited an immunity to the colicins produced by all the plasmids they carried and also produced colicins corresponding to all the plasmids they carried. An incompatibility was observed among the plasmids. In doubly colicinogenic cells where the presence of two plasmids was established, Col E2 was lost more frequently than Col E3. In triply colicinogenic cells, Col E1, Col E2, and Col E3 were lost, with Col E3 being lost least frequently. A significant reduction in the acquisition of a conjugationally transferred Col E1 plasmid by cells colicinogenic for Col E1 was demonstrated.     

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3.

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.     

The immunity genes of colicins E2 and E8 are closely related

We have determined the nucleotide sequence of the newly characterized colicin E8imm gene which exists in tandem with the colicin E3imm gene in the: ColE3-CA38 plasmid. Comparison of these immunity structures reveals considerable sequence divergence) but the ColE8imm gene is markedly homologous to the colicin E2imm gene from the ColE2-P9 plasmid.Issued as NRCC no. 23586 and as CBRI no. 1480.     

Colicin E1 in planar lipid bilayers

The channel formed by the C-terminal domain of colicin E1 in planar lipid bilayers has proven to be more complex than one might have guessed for such a simple system. The protein undergoes a pH-dependent rearrangement which transforms it from a water soluble form to a much different membrane bound form. There are at least two bound states which don't form a channel. The process by which the channel opens and closes is regulated by the pH and the transmembrane voltage. The voltage is probably sensed by at least 3 (and more likely 4 or more) lysine residues which must be driven through the field to open the channel. The process appears to be hindered by particular carboxyl groups when they are in the unprotonated state. The open channel has several substates and several superstates. Very large positive voltage catalyzes a transition of the open channel to an inactivated state, and may be able to drive the channel-forming region of the protein across the membrane. Little is known about the structure of any of these states, but the open channel is large enough to allow NAD to traverse the membrane and appears to be formed by one colicin molecule. This single polypeptide mimics many of the properties found in channels of mammalian cell membranes, but it may prove more relevant as a model for the transport of proteins across membranes. The comparative ease with which the protein can be manipulated chemically and genetically, along with the complexity of its behavior, promises to keep several laboratories busy for some time.     

Isolation and Characterization of a Mutant Colicin E2

Escherichia coli K-12 colicinogenic for Col E2 yielded a mutant, SK95, that carries a nonsense mutation in the colicin structural gene. A derivative of SK95 that carries an as yet unidentified suppressor mutation produces a colicin E2 that is temperature sensitive (TS). This mutant colicin kills sensitive cells at low temperature but not at high temperature; the colicin adsorbs to cells at high temperature but does not kill them unless the temperature is lowered. Unlike normal colicin E2, which adsorbs rapidly to cells, TS colicin E2 adsorbs slowly over a period of several hours. The biochemical target of colicin E2 is deoxyribonucleic acid (DNA). When acid solubilization of DNA was compared in cells treated with either TS or normal colicin E2, striking differences were observed. Cell killing and acid solubilization of DNA by colicin E2 were shown to be separable events under certain conditions. The results are discussed in relation to the mechanism of action of colicin E2.     

Colicin E1 export inSalmonella typhimurium wild-type and lipopolysaccharide mutants

Colicin export was studied in differentSalmonella typhimurium strains lacking the O-antigen repeating units (O⁻) and different strains with different chemotypes for the lipopolysaccharide core, as well as the wild-type strain (O⁺) to determine the role of lipopolysaccharide length on colicin E1 export. While the lipopolysaccharide length influences the levels of external hemolytic activity inS. typhimurium, no effect was detected on colicin E1 export.     

Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein

Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6. The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees. The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day. Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit.     

Genetic analysis of the functional relationship between colicin E3 and its immunity protein.

Partial deletions in the immunity gene of the colicin E3 operon were used to study possible functions of the immunity protein besides protection against exogenous colicin. Nuclease BAL-31 was used to create a series of carboxyl-terminal deletions of the immunity gene. Mutants displaying lowered immunity against exogenous colicin were found, and six that had reduced but detectable levels of immunity were chosen for further analysis. DNA sequence analysis of the deletions showed that all six terminated within the last five codons of the immunity gene. The wild-type immunity gene was replaced by each of the six mutated immunity genes in a plasmid containing an otherwise functional colicin E3 operon. Transformants containing the resulting plasmids produced smaller colonies on solid medium and grew more slowly in liquid culture than transformants carrying the wild-type colicin and immunity genes. This result suggested that immunity protein was required to protect the cell against endogenous colicin E3. This idea was confirmed in experiments in which the colicin E3 and immunity genes were independently cloned on two compatible plasmid vectors.     

Stabilization of Colicin E2 by Bovine Serum Albumin

Colicin E2 was partially purified from Escherichia coli W3110. This preparation was remarkably stabilized by bovine serum albumin in a solution at neutral pH, as shown by dilution experiments and tests on heat stability of colicin. One killing unit of colicin E2 was estimated to correspond to one molecule of colicin E2, on the assumption of a molecular weight of 60,000.     

Synthesis of Colicin E1 in a Cell-Free System

Colicin E1 was synthesized in a cell-free system. The in vitro synthesis was found to be dependent on the Col E1 DNA concentration and was not enhanced by the addition of mitomycin C.     

Colicin E2 is still in contact with its receptor and import machinery when its nuclease domain enters the cytoplasm

Colicins reach their targets in susceptible Escherichia coli strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Colicin E2 (ColE2) is a cytotoxic protein that recognizes the outer membrane receptor BtuB. After gaining access to the cytoplasmic membrane of sensitive Escherichia coli cells, ColE2 enters the cytoplasm to cleave DNA. After binding to BtuB, ColE2 interacts with the Tol system to reach its target. However, it is not known if the entire colicin or only the nuclease domain of ColE2 enters the cell. Here I show that preincubation of ColE2 with Escherichia coli cells prevents binding and translocation of pore-forming colicins of group A but not of group B. This inhibition persisted even when cells were incubated with ColE2 for 30 min before the addition of pore-forming colicins, indicating that ColE2 releases neither its receptor nor its translocation machinery when its nuclease domain enters the cells. These competition experiments enabled me to estimate the time required for ColE2 binding to its receptor and translocation.