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At least 19 recordsLinked to original sources
PubMed2004

Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A-porin complexes.

Release of colicin A was studied in Escherichia coli cells that differed in expressing the colicin A lysis protein (Cal). Pools of released and unreleased colicin A were harvested throughout colicin A induction. The amount of colicin A in each pool varied with the time of induction, allowing the definition of two sequential steps in colicin A release, one of which was dependent on Cal. Each step of colicin A release was differently affected in cells containing Cal mutants in which the N-terminal cysteine residue was substituted by either proline or threonine, preventing them from being acylated and matured. These Cal mutants were only observed in degP cells, indicating that the DegP protease cleaved the unacylated precursor of Cal. Cal was found in the insoluble fraction of the pools of released and unreleased colicin A together with the hetero-oligomers of colicin A and porins (colicins Au). The biogenesis of colicins Au was studied in temperature-sensitive secA and secY strains and found to be Sec-independent, indicating that they are formed by newly synthesized colicin A binding to mature porins already incorporated in the outer membrane. Cal is a lipoprotein similar to VirB7, a constituent of the type IV secretion system. It would interact with colicins Au to constitute the colicin A export machinery.

Adenosine Triphosphatases↗
PubMed1976

Sensitization of colicin K-treated bacteria by sodium dodecyl sulfate: presence of free colicin in colicin K-treated cultures of Escherichia coli.

A method of assaying colicin K is described. It makes use of two properties of sodium dodecyl sulfate to protect bacteria against colicin action and to dissolve those bacteria on which colicin K had started its action. By this method, the kinetics of bacterial killing by colicin K have been measured directly in the treated culture without intervening dilution. The kinetics are exponential with time and are a function of the colicin multiplicity, as described previously, but do not reach a final plateau. At any time during colicin treatment, free colicin is found in the medium. Procedures that eliminate or destroy this free colicin, such as centrifugation and resuspension of the treated bacteria in a fresh medium, or addition of trypsin or sodium dodecyl sulfate to the treated culture stop bacterial killing.

Colicins↗
PubMed1980

Escherichia coli K317, formerly used to define colicin group E2, produces colicin E7, is immune to colicin E2, and carries a bacteriophage-restricting conjugative plasmid.

Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317. This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6. Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9. A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins. We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317. Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9. We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids.

Colicins↗
PubMed1977

Comparison of colicins B-K260 and D-CA23: purification and characterization of the colicins and examination of colicin immunity in the producing strains.

Colicins B-K260 and D-CA23 were purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography and were compared with respect to a number of physical and chemical properties. Both colicins were shown to be proteins and were found to have similar molecular weights, isoelectric points and amino acid compositions. The two colicins also have substantial antigenic similarities but are distinguished by the presence of non-cross-reacting antigens and by differences in stability and in sensitivity to heat and reducing conditions. In addition, strains of Escherichia coli K-12 producing colicins B-K260 and D-CA23 are not cross-immune. The similarities noted between the two colicins are compatible with their use of a common cell surface receptor while having different modes of action.

Amino Acids↗
PubMed1998

The tip of the hydrophobic hairpin of colicin U is dispensable for colicin U activity but is important for interaction with the immunity protein.

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575-582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575-582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.

Amino Acid Sequence↗
PubMed1989

Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D.

The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74,683 D and that the immunity protein had a molecular weight of 10,057 D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.

Amino Acid Sequence↗
PubMed1997

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.

Amino Acid Sequence↗
PubMed1968

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.

Adenosine Triphosphate↗
PubMed2000

Competition by allelopathy proceeds in traveling waves: colicin-immune strain aids colicin-sensitive strain.

Producing toxic chemicals to suppress both the growth and survivorship of local competitors is called allelopathy; some strains of the bacteria Escherichia coli produce a toxin (named colicin) which may kill colicin-sensitive neighbors while they themselves are immune. In a previous paper, the competitive outcome between colicin-producing and colicin-sensitive strains was shown to differ between a spatially structured and a completely mixed population. In this paper, we analyze the role of a third, "colicin-immune," strain, which does not produce colicin but is immune to it. Without spatial structure, the colicin-immune strain suppresses the colicin-producing strain and enables the colicin-sensitive strain to win. In a spatially structured population, modeled as a reaction-diffusion system, we examine the speed of boundaries between areas dominated by different strains in traveling waves and the events after the collision of two such boundaries. The colicin-immune strain passes through the area dominated by the colicin-sensitive strain and drives the colicin-producing strain to extinction. Subsequently the colicin-sensitive strain occupies the whole population.

Alleles↗
PubMed1997

The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step.

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.

Alkaline Phosphatase↗
PubMed1994

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake.

The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens, or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.

Bacterial Proteins↗
PubMed2004

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import.

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.

Bacterial Outer Membrane Proteins↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement.

Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.

Bacterial Outer Membrane Proteins↗
PubMed1987

Nucleotide sequencing of the structural gene for colicin N reveals homology between the catalytic, C-terminal domains of colicins A and N.

An 1800 bp fragment of DNA from a natural ColN plasmid (pCHAP4) encompassing the colicin N structural gene (cna) and its regulatory region was subjected to nucleotide sequencing and deletion analysis. The region of DNA immediately upstream from cna contains two tandemly-arranged and overlapping potential LexA binding sites (SOS boxes), in line with the previous demonstration that cna expression is repressed by LexA protein. Deletion of the LexA binding site allowed efficient transcription of cna from an upstream lacZ promoter, whereas its presence reduced lacZ-promoted cna expression to varying extents depending on the proximity of lacZp and the SOS boxes. The molecular weight of colicin N, as deduced from the nucleotide sequence, is 41,696, which is close to the experimentally determined molecular weight of 39,000. Colicin N has a glycine-rich amino terminus similar to that found in many other colicins. Part of the glycine-rich domain of colicin N could be replaced by an unrelated sequence devoid of glycine residues without affecting either colicin release or activity. The carboxy-terminal half of colicin N exhibits significant homology to the C-terminus of colicin A. The latter colicin forms pores in the cytoplasmic membrane of Escherichia coli, thereby depolarizing the membrane and causing cell death. The C-terminus of colicin A is endowed with this catalytic activity. Although colicin N was previously found to cause lysis of Escherichia coli cells, a more detailed investigation revealed that it too depolarizes the Escherichia coli cytoplasmic membrane and that lysis is a secondary effect.

Amino Acid Sequence↗
PubMed1975

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

By using each of the available colicins, we have isolated a large number of colicin-resistant mutants. They included both receptor and tolerant mutants and each was screened for cross-resistance to all other colicins. On the basis of the cross-resistance of these mutants it was possible to place known colicins into two mutually exclusive groups, group A and group B. Mutants selected as resistant to colicins of group A may or may not be cross-resistant to other colicins of group A, BUT Are never resistant to colicins of group B. The reverse also applies. The mutants isolated as resistant to colicins of group A (A, E1, E2, E3, K, L, N, S4, and X) have been divided into 21 phenotypic classes on the basis of their colicin resistance patterns. These include most of the tolerant and receptor mutants previously isolated, some of which were previously shown to also have an increased sensitivity to certain antibiotics and detergents. Type strains from each of the phenotypic classes were therefore tested for sensitivity to a range of antibiotics, detergents, and surfactants that included all those previously used. With these new data, it has been possible to speculate informatively on the mode of action of the different colicins. We have confirmed the position of previously isolated mutations on the Escherichia coli K-12 genetic map, and located approximately the loci conferring colicin resistance in some of the newly isolated mutants.

Anti-Bacterial Agents↗
PubMed1989

Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13.

The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined. Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene. The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities. On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons. The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein. The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13. Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13).

Amino Acid Sequence↗
PubMed1997

The TolA protein interacts with colicin E1 differently than with other group A colicins.

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.

Amino Acid Sequence↗