Strong function-related homology between the pore-forming colicins K and 5.

Sequence determination of the Escherichia coli colicin K determinant revealed identity with the E. coli colicin 5 determinant in the immunity and lysis proteins, strong homologies in the pore-forming region (93.7%) and the Tsx receptor-binding region (77%) of the colicins, and low levels of homology (20.3%) in the N-terminal region of the colicins. This latter region is responsible for the Tol-dependent uptake of colicin K and the Ton-dependent uptake of colicin 5 in the respective colicins. During evolution, the DNA encoding colicin activity and binding to the Tsx receptor was apparently recombined with two different DNA fragments that determined different uptake routes, leading to the differences observed in colicin K and colicin 5 import.     

Periplasmic chaperone FkpA is essential for imported colicin M toxicity

Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 10⁴-fold dilutions killed fkpA ⁺ cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS 1 insertion in the peptidyl-prolyl- cis-trans -isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro ; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.     

A physiological role for DNA supercoiling in the anaerobic regulation of colicin gene expression

Summary The mechanism of anaerobic regulation of synthesis of colicins E1, E2, E3, K and D was studied. It was found that anaerobiosis significantly increases expression of the genes for colicins E1, E2, E3, K, and D. Experiments with novobiocin (a DNA gyrase inhibitor) showed that colicin synthesis in minicells and derepressed colicin synthesis in cells are dramatically reduced by relaxation of DNA supercoiling. A good correlation was observed between the levels of colicin synthesis and plasmid DNA supercoiling and the degree of aeration of the cultures. Thus, the regulation of colicin gene expression in response to a change in aeration appears to be mediated by environmentally induced variations in DNA supercoiling.     

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.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

Summary After transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.     

Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K

Escherichia coli B, induced for β-galactoside permease, can accumulate thiomethyl-β-galactoside in the cell even at 0 °C. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-β-galactoside uptake. Inhibition by colicin K is, however, seen at 0 °C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration of exposure. There is a transition point at around 21 °C in Arrhenius plots of this colicin K activation reaction.If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-β-galactoside uptake after returning the colicin K-cell complex to 0 °C and removal of the inhibitors) is prevented.These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.     

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.     

Domains of colicin M involved in uptake and activity

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the Tong, ExbB and ExbD proteins. These colicins contain a sequence, called the Tong box, which has been implicated in transport via Tong. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with Tong may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This Colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193–197 and 223–252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the Tong box at the N-terminal end of colicin M must be involved in colicin uptake via Tong across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.     

Detection of induced synthesis of colicin E9 using ColE9p::gfpmut2 based reporter system

The majority of colicin operons are regulated by an SOS response inducible promoter (SOS promoter), located at upstream of the colicin operons. Therefore, colicin synthesis is induced by DNA damaging agents like mitomycin C (MMC) because the resulting DNA damage switches on the SOS response in bacteria. In this study, we have described the strategy for fusion of the SOS promoter of the colicin E9 operon (ColE9p) with a promoterless green fluorescent reporter gene (gfpmut2). We observed that the ColE9p–gfpmut2 is inducible by MMC which confirmed that the ColE9p–gfpmut2 is sensitive to SOS response inducing agents. The data implies that the ColE9p–gfpmut2 based reporter system is suitable for monitoring the ColE9 synthesis induced by SOS response inducing agents including antibiotics. Using green fluorescent protein expression from the ColE9p–gfpmut2 as an indicator of ColE9 synthesis; we have investigated, first time, the inducing effects of cephalexin antibiotic on ColE9 synthesis. Our data demonstrated that the cephalexin has potential to induce ColE9 synthesis from E. coli JM83 host cells albeit the level of this induction is very low hence its detection required a highly sensitive method.     

Energy requirement for the initiation of colicin action in Escherichia coli

1. Starved cells of a strain of Escherichia coli and its mutant uncA, treated with colicin K, E2 or E3, remained fully rescuable upon trypsin treatment (stage I in colicin action). The transition to stage II in colicin action (cells no longer rescuable by trypsin) was promoted by the addition of either glucose or d-lactate.2. Aerobically glucose-grown cells of the normal strain were irreversibly killed by colicin K, E2 or E3 under anaerobic conditions, while similarly treated cells of its mutant uncA remained fully rescuable. The stage I-stage II transition in colicin action was blocked in normal cells under anaerobic conditions when succinate was the sole carbon source.3. Arsenate alone had little effect on the progression of the stage I-stage II transition in normal cells, treated with colicin K. However, this transition was abolished in the presence of both arsenate and anaerobic conditions.4. The initiation of colicin action could be coupled to the anaerobic electron transfer systems formate dehydrogenase-nitrate reductase and α-glycerophosphate dehydrogenase-fumarate reductase.5. These results indicate that an energized state of the cytoplasmic membrane is required for the initiation of colicin action and that no high-energy phosphorylated compounds are necessary.     

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.     

A colicin M derivative containing the lipoprotein signal sequence is secreted and renders the colicin M target accessible from inside the cells

Colicin M is only released in very low amounts by cells harbouring this plasmid encoded colicin, due to the lack of a release (lysis) protein. A fusion gene (lpp'cma) was constructed which determined two proteins: Lpp'-Cma composed of the signal sequence of the murein lipoprotein (Lpp) and colicin M (Cma), and unaltered colicin M. Cells expressing the fusion gene released 50% of the total colicin M into the culture medium, compared to 1% found in the medium of cells synthesizing only colicin M. The release resulted from partial cell lysis caused by colicin M since a colicin M tolerant strain remained unaffected. Lpp'-Cma thus mimics phenotypically the action of colicin release proteins but displays a different lysis mechanism. In strains defective in components of the colicin M uptake system, Lpp'-Cma caused lysis as effectively as in uptake proficient strains. Apparently, Lpp'-Cma renders the colicin M target site accessible from inside the cell which stands in contrast to the action of colicin M which is only bactericidal when provided from outside.Abbreviation bp base pairs     

Replication of colicin E1 plasmid DNA in vivo requires no plasmid-encoded proteins.

A derivative of bacteriophage lambda containing a colicin E1 plasmid replicon was constructed by recombinant DNA techniques. This phage, lambdacol100, has two functional modes of DNA replication; it can replicate via either plasmid or phage replication systems. lambdacol100 has been used to introduce the colicin E1 plasmid replicon into Escherichia coli previously treated with chloramphenicol to block protein synthesis. Under these conditions, lambdacol100 DNA is replicated normally as a colicin E1 plasmid. This suggests that colicin E1 plasmid replication in vivo does not require any plasmid-encoded proteins.