Characterization of the cea gene of the ColE7 plasmid.

Summary The complete nucleotide sequence (1731 nucleotides) of the gene encoding colicin E7 (cea) of plasmid CoIE7-K317 was determined. This sequence encoded a deduced polypeptide of 576 amino acids of molecular weight 61349 Da. Comparison of the nucleotide and amino acid sequences ofcea E7 with those of other E-group colicins revealed that colicin E7 was closely related to colicin E2, both in gene sequence and in predicted secondary structure of the deduced protein. Judging from the results of cross-immunity tests, we postulated that CoIE7 is probably a proximate ancestor of Co1E2 and Co1E8. Based on results from colicin production tests on cells harboring a 5 end deleted form of thecea E7 gene, we propose, that a previously unknown, non-inducible promoter may be involved in regulation of the constitutive expression of thecea E7 gene.     

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.     

Analysis of a cloned colicin Ib gene: complete nucleotide sequence and implications for regulation of expression.

The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed.     

Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein

A major group of colicins comprises molecules that possess nuclease activity and kill sensitive cells by cleaving RNA or DNA. Recent data open the possibility that the tRNase colicin D, the rRNase colicin E3 and the DNase colicin E7 undergo proteolytic processing, such that only the C-terminal domain of the molecule, carrying the nuclease activity, enters the cytoplasm. The proteases responsible for the proteolytic processing remain unidentified. In the case of colicin D, the characterization of a colicin D-resistant mutant shows that the inner membrane protease LepB is involved in colicin D toxicity, but is not solely responsible for the cleavage of colicin D. The lepB mutant resistant to colicin D remains sensitive to other colicins tested (B, E1, E3 and E2), and the mutant protease retains activity towards its normal substrates. The cleavage of colicin D observed in vitro releases a C-terminal fragment retaining tRNase activity, and occurs in a region of the amino acid sequence that is conserved in other nuclease colicins, suggesting that they may also require a processing step for their cytotoxicity. The immunity proteins of both colicins D and E3 appear to have a dual role, protecting the colicin molecule against proteolytic cleavage and inhibiting the nuclease activity of the colicin. The possibility that processing is an essential step common to cell killing by all nuclease colicins, and that the immunity protein must be removed from the colicin prior to processing, is discussed.     

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.     

Mutation analysis of the receptor for colicins E1–E7. A pilot study

Thirty eight mutant clones of the colicin indicator strainEscherichia coli K 12 ROW, selected by their insensitivity to any of the colicins El–E7, were isolated. Comparison of their sensitivity-resistance patterns to colicins El–E7 enabled us to draw a rough preliminary map of the receptor for E colicins. In this receptor, the highly specific binding site for colicin El partially overlaps with the domain shared by all colicins E2 through E7. A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7. Using colicin assay experiments, the binding capacity of coliein E receptor mutants could be estimated. A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found. Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity—resistance is not a qualitative but a quantitative marker.     

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 74683D and that the immunity protein had a molecular weight of 10057D. 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.     

Change of thermal stability of colicin E7 triggered by acidic pH suggests the existence of unfolded intermediate during the membrane-translocation phase

Purified colicin E7 was analyzed by CD spectrum and gel filtration chromatography in a mimicking membrane-translocation phase. It was found that the CD spectra of colicin E7 at pH 7 and pH 2.5 were similar. Although the melting temperature of the protein shifted from 54.5°C to 34°C at low pH, the thermal denaturation curves of colicin E7 at different pH conditions still fit a two-state model. These experimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein melting. In contrast to the minor change in secondary structure at different pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from the partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating that this protein complex is indeed formed by aggregation of the monomeric colicins. Most interestingly, the aggregated colicins still perform in vivo bacteriocidal activity. We suggest that in a partial unfolding state the colicin is prepared for binding to the specific targets for translocation through the membrane. However, in the absence of specific targets in vitro these unfold intermediates may therefore aggregate into the multimeric form of colicins. Proteins 32:17–25, 1998. © 1998 Wiley-Liss, Inc.     

Genetics of colicin E susceptibility inCitrobacter freundii

The insensitivity ofCitrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants ofC. freundii also lost their colicin E1 receptor function. Sensitivity to colicin E1 can be induced by F′gal ⁺ tol ⁺ plasmids, thetol A⁺ gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by theE. coli F′14bfe ⁺ plasmid, which is also able to enhance notably the receptor capacity for colicin E1. Thebfe ⁺ gene product ofE. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants ofC. freundii. All results show that there is a remarkable difference between theE. coli bfe ⁺ gene product and thebfe ⁺ gene product ofC. freundii and also between thetol A⁺ gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F′14bfe ⁺ plasmid.     

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.     

Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli

Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. We first produced KT fused to a signal sequence for periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.Colicins are bacterial toxins produced by Escherichia coli strains and are active against E. coli or related strains (17). These bacterial antibiotic toxins play an important role in the E. coli colonization of environmental niches, including the mammal gastrointestinal tract (25, 32, 49, 50). The classification of colicins is based on differences in the mechanisms of action, such as pore formation (colicins A, B, E1, K, Ia, N, 5, etc.), degradation of nucleic acids (including DNases [colicins E2, E7, and E9], 16S RNases [colicins E3, E4, and E6], or tRNases [colicins D and E5]), or degradation of lipid II (colicin M) (17, 34). Colicins are also categorized depending on their import machines: colicins using the Tol proteins are classified as group A (colicins A, E1 to E9, K, N, etc.), whereas colicins using the ExbBD-TonB proteins are classified as group B (colicins B, D, Ia, M, 5, etc.). However, the transport across the periplasm is only one of the three steps of the mechanism of action. Colicins bind to an outer membrane receptor and are translocated through the outer membrane and the periplasm (14, 35, 55, 56). Finally, the C-terminal domain (responsible for the activity) is translocated to its final destination (inner membrane or cytoplasm) depending on its mechanism of action. Colicins are divided into three different structural and functional domains that correspond to the three steps of the mechanism of action: the N-terminal domain is required for translocation, the central domain is involved in receptor binding, and the C-terminal domain carries the activity (4, 5). During the translocation step, the N-terminal domain of the colicin interacts with components of the import machine: colicins A, E1, and N interact with the TolA protein; colicins A, E3, E7, and E9 interact with the TolB protein; and colicins A and E3 interact with TolR (6, 12, 13, 15, 21, 23, 26, 27, 30, 39, 48, 54). In some cases, the domains of the Tol proteins involved in colicin binding have been identified. Reciprocally, the regions of colicins in interaction with the Tol proteins have been delineated. In colicin A, the TolA binding sequence (ABS) is contained within residues 37 to 98 (13, 30), in which a SYNT motif (residues 57 to 60) has been shown to be essential for TolA binding (18, 46). The TolB box and the TolR binding sequences have also been identified in colicin A (27, 30). The TolB box is well conserved within TolB-dependent colicins, including colicins A and E2 to E9, and is composed of residues DG[T,S]GWSSE (12, 13). These residues form a loop penetrating within the TolB beta-propeller (39, 57), mimicking the TolB-Pal interaction (9, 10). Interestingly, the Tol-dependent, pore-forming colicin K does not possess a TolB box (see Fig. ​Fig.1A),1A), raising the hypothesis that its translocation might be TolB independent or that colicin K interacts with TolB differently than do other TolB-dependent colicins. In this study, we tested the Tol requirements for colicin K translocation and showed that colicin K requires the TolA, TolB, TolQ, and TolR proteins. Production of the N-terminal domain of colicin K in the periplasm of wild-type (WT) cells induces specific tol defects and tolerance to Tol-dependent colicins and bacteriophage, suggesting that the colicin K N-terminal domain binds and titrates the Tol proteins. Further in vivo coimmunoprecipitation and in vitro pulldown experiments demonstrated interactions between the colicin K N-terminal domain and the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between a colicin translocation domain and the fourth component of the Tol complex, the TolQ protein.Open in a separate windowFIG. 1.In the absence of an identifiable TolB-binding sequence, colicin K translocation is TolB dependent. (A) Sequence alignment of colicin K and three TolB-dependent colicins (A, E2, and E9). Conserved residues are indicated by red letters. The characterized TolB binding sequence is indicated by the green box (defined in references 12 and 27). (B) Colicin spot assays using serial dilutions of colicins A (TolB dependent), E1 (TolB independent), and K on a wild-type (WT) strain and its tolB derivative (from left to right, 100, 10, 1, and 0.1 ng of colicins have been spotted, respectively).     

Molecular Characterization of the Klebicin B Plasmid of Klebsiella pneumoniae

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.