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Colicin Biology

Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France,¹ Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892,² Department of Biology Area 10, P.O. Box 373, University of York, York YO10 5YW, United Kingdom,³ 301 Althouse Laboratory, Department of Biochemistry and Molecular Biology, Eberly College of Science, Pennsylvania State University, University Park, Pennsylvania 16802,⁴ Department of Biology, University of Massachusetts at Amherst, Amherst, Massachusetts 01003,⁵ Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461⁶

SUMMARY

INTRODUCTION

COLICIN SYNTHESIS

Colicinogenic Plasmids

Colicin Operons: Gene Organization and Regulation

Colicin Expression

Lethality of Colicin Production

COLICIN RELEASE

Sequence, Synthesis, and Localization of the Colicin Lysis Proteins

Functions of Colicin Lysis Proteins

Colicin release.

Quasilysis.

Modifications of cell envelope structure.

Activation of OmpLA, the outer membrane phospholipase A.

Death of the host cell.

Regulator of expression of the colicin operon.

Structure-Function Relationships of Colicin Lysis Proteins

Mechanism of Action of Colicin Lysis Proteins

STRUCTURAL ORGANIZATION OF COLICINS

Domain Organization of Colicins

Specific Functions of Colicin Domains

Three-Dimensional Structures of Colicins

COLICIN RECEPTION

Recognition of the Receptor at the Bacterial Cell Surface

Binding to the Receptor

Competition with the Natural Ligand

Colicin Cleavage at the Cell Surface

Energy Requirements for Receptor Binding

Release of Immunity

Reception of Bacteriophages

COLICIN IMPORT

TRANSLOCATION THROUGH THE OUTER MEMBRANE

Tol-Dependent Colicins

TonB-Dependent Colicins

TRANSIT THROUGH THE PERIPLASM

General Principles of Colicin Transit

Tol-Dependent Colicins

The Tol system. (i) Identification.

(ii) Regulation.

(iii) Localization and topology.

(iv) Three-dimensional structures.

(v) Interaction network.

(vi) Physiological functions.

Interactions between Tol subunits and colicin translocation domains.

(i) Interaction with the TolA protein.

(ii) Interaction with the TolB protein.

(iii) Interaction with the TolR and TolQ proteins.

Definition of Tol boxes.

(i) TolA binding sequence.

(ii) TolB binding sequence.

(iii) TolR binding sequence.

Structural information on Tol-dependent translocation.

Hierarchy of contacts during transit.

Kinetics of translocation.

TonB-Dependent Colicins

The TonB protein: physiological function and its role in colicin translocation. (i) The TonB amino terminus.

(ii) The proline-rich region.

(iii) The TonB carboxy terminus.

(iv) The carboxy terminus in vitro.

(v) What do the crystal/NMR structures represent?

(vi) The carboxy terminus in vivo.

(vii) Models for TonB energy transduction.

New thoughts on an old protein.

The ExbB and ExbD proteins: physiological function and their role in colicin transit.

Cross-Complementation between Tol and TonB Systems

Energy Dependence for Translocation

Speculative Models for Colicin Translocation

Translocation of Phage DNA

Similarities between colicin and phage DNA translocation.

Interaction between Tol subunits and minor capsid phage translocation domains: a TolA box?

Energy requirements for phage DNA uptake.

Speculative models for phage DNA translocation.

COLICIN ACTIVITIES

Pore-Forming Colicins

The closed channel.

The open channel.

Channel gating and translocation.

Immunity to pore-forming colicin.

(i) Immunity proteins interact with the pore-forming domain in the inner membrane.

(ii) Immune process involves intramembrane association.

Enzymatic Colicins

Overview of nuclease colicins and their immunity proteins.

Nuclease transport across the inner membrane.

(i) Colicin nucleases associate with anionic phospholipids.

(ii) Translocation across the IM and refolding.

(iii) Processing of colicin nuclease domains.

Structural biology of colicin nucleases and their modes of action.

(i) Colicin DNases.

(ii) RNase colicins.

Nuclease-specific immunity proteins.

(i) tRNase-specific immunity proteins block the enzyme active site.

(ii) DNase- and rRNase-specific immunity proteins are high-affinity exosite inhibitors.

Colicin M.

COLICIN-LIKE BACTERIOCINS FROM OTHER BACTERIAL GENERA

Pesticins

Klebicins or Klebocins

Pyocins

Lumicins

Megacins

COLICINS AND PHAGES AS LABORATORY TOOLS

Bacterial Containment

Biosensing of Genotoxic Compounds

Improvement in Protein Purification

Production of Outer Membrane Vesicles

Screening of Biological Processes

Phage Display and Role of the g3p Protein

COLICIN EVOLUTION AND ECOLOGY

Two-Step Process in Colicin Evolution

Role of Colicins in Promoting Microbial Diversity

CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Colicins are proteins produced by some strains of Escherichia coli that are lethal for related strains of E. coli. The first colicin was identified by Gratia in 1925 as a heat-labile product present in cultures of E. coli V and toxic for E. coli (235). Further on, numerous colicins produced by different strains of the enteric group of bacteria (E. coli, Shigella, and Citrobacter) were characterized. The name colicin was coined by Gratia and Fredericq in 1946, who demonstrated their protein nature and the specificity of their activity spectra (236). Afterwards, the term bacteriocin was introduced to designate toxic proteins produced by a given strain of bacteria and active against related species but not on the producing cells (296). By analogy with colicins, the new families of bacteriocins carry the name of the producing species of bacteria followed by the suffix -cin. Thus, pyocins from Pseudomonas pyogenes strains, cloacins from Enterobacter cloacae, marcescins from Serratia marcescens, megacins from Bacillus megaterium, etc., have been identified. A nomenclature using the genus name in place of the species name of the producing bacteria has been proposed by Fredericq to avoid redundancies (209). According to the nomenclature, the bacteriocins produced by Pasteurella pestis and by Yersinia pestis would not have both been called pesticins (175, 547) but would have been called pasteurellacins and yersiniacins. Fredericq's advice has not been followed, perhaps in order to retain the word colicin in place of escherichiacin. In our days, the meaning of the word bacteriocin has changed, since it is now used mainly to designate antibiotic peptides produced by gram-positive bacteria and active on a wide range of bacteria. The producers of these toxic peptides, as the strains producing protein bacteriocins, possess a specific immunity mechanism to protect themselves against their own bacteriocin (reviewed in references 128 and 162). Confusions in the nomenclature must be heeded, although they are as old as colicin studies: the first identified colicin, colicin V, is now classified among the microcins but is still called colicin (225, 687). The microcins are a family of low-molecular-weight antibiotics produced by Enterobacteriaceae and are active against phylogenetically related microbial strains (reviewed in references 11 and 294).

The narrow target range of colicins has been shown by Fredericq to be due to the presence of specific receptors at the surface of the sensitive strains on which colicin binds before killing (208). Mutation of the receptor can lead to the loss of sensitivity to the corresponding colicin. Mutants that are resistant to each colicin have been isolated and used as the basis to name each colicin by the alphabet letter used, at the time, to designate the receptor to which it binds. When more than one colicin binds to the same receptor, they are designated by the alphabet letter of the receptor followed by a number, as, for instance, the nine colicins E: E1 to E9. The receptors have been shown to be outer membrane (OM) proteins that allow the entry of specific nutrients such as nucleosides, siderophores, and vitamins (103, 104, 158). BtuB, the receptor of vitamin B₁₂, of the nine colicins E, and of the phage BF23, was the first colicin receptor purified by Sabet and Schnaitman in 1973 (567).

Interest in colicin studies started up in earnest with the work of Jacob et al. in 1952 (297). Using colicin E1 produced by E. coli ML30, those authors demonstrated that (i) the production of colicin by colicinogenic E. coli cells is induced by SOS agents, as is seen with lysogenic phages, and is lethal for producing cells; (ii) the produced colicin is released into the medium late after synthesis (later shown not to be the case for all colicins); (iii) colicin kills sensitive cells according to single-hit kinetics; and (iv) colicin is not active against the producing bacteria due to the presence of a specific antagonist protein called the immunity protein. They compared colicins to bacteriophages, with which they share various properties including specificity of the activity spectra, binding on specific receptors (some of which are common for a given colicin and phage), single-hit mode of action, specific immunity, and lethal production after treatments with mutagenic agents (297, 430). This major contribution triggered numerous studies on the mode of action of colicins.

In 1963, Nomura demonstrated that the various colicins have different modes of action: colicins E1 and K inhibit all macromolecular synthesis without arrest of respiration, colicin E2 causes DNA breakdown, and colicin E3 stops protein synthesis (484). In every case, the colicin lethal action appears to be reversed by treatment with trypsin during a given time period (486). To explain this rescue, a model was proposed in which colicin remains on the bacterial surface at the receptor site and kills the cell from there, in a single-hit process, by sending a signal that is not lethal until it is amplified and reaches its target (484, 485). Rescue from colicin bound to the receptor by trypsin takes place during transmission of the signal, seeing as the time available for rescue is prolonged by energy poisons such as azide and 2,4-dinitrophenol (486). In subsequent work, rescue of cells treated with colicin was obtained with various agents that inactivate free colicin, such as sodium dodecyl sulfate (SDS), salts, and antibodies to colicins (80, 100, 437, 438). It has been proposed that colicin binding is reversible and that rescue might be obtained as long as colicin adsorption does not reach an irreversible state (80, 100, 131a, 437, 583). However, according to the agent used, rescue is obtained with different kinetics, as would be expected to occur if particular agents acted at particular steps of colicin action (100). One of the irreversible events of the colicin lethal action was thought to be the activation of OmpLA, the outer membrane phospholipase A of sensitive cells (100, 101). Thus, one stage of colicin action does not provoke cellular damage as cells are rescued by trypsin treatment, while damage occurs in a second stage (523).

Mutations of the cellular components required for colicin bound on its receptor to transmit its signal have thus been researched. Insensitive mutants that nevertheless possess colicin-specific receptors were isolated in 1967 and were called either tolerant (475, 487) or refractory (266) to distinguish them from the resistant mutants described previously. Different tolerant mutants were afterwards characterized and have been shown to map to either the tol or the tonB gene, defining two machineries used by colicins to enter into the cell, a given colicin always using the same pathway to reach its target. That allowed a classification of colicins into two groups, groups A and B, based on cross-resistance (133, 134). Group A comprises colicins that are translocated by the Tol system, such as colicins A, E1 to E9, K, L, N, S4, U, and Y, while group B comprises colicins that use the TonB system, such as colicins B, D, H, Ia, Ib, M, 5, and 10. It was later shown that the A and B groups are also distinguished by their mechanism of release from the producing cell. In general, group A colicins are encoded by small plasmids and are released into the medium, whereas group B colicins are encoded by large plasmids and are not secreted. However, some colicins might belong to one group and share homologies with colicins of the other group. That is the case of colicins 5 and 10 (515, 516).

It took time to demonstrate that colicin itself is translocated through the cell envelope rather than any putative constituents that play an intermediate role during its action. The demonstration in 1971 that colicin E3 is a specific RNase that makes one cut in the 16S rRNA gene (35, 45, 581) significantly changed the model of colicin action. Further on, the endonuclease activity of numerous colicins was demonstrated, with each one specifically cleaving a particular nucleic acid at a precise site. Colicins E2, E7, E8, and E9 cleave DNA (108, 574, 628), and colicins E3, E4, and E6 and cloacin DF13 hydrolyze rRNA (35, 45, 138), while colicins D and E5 cleave tRNA (491, 629). The killing action of colicins that stop cell metabolism has been more difficult to elucidate. In 1978, Finkelstein's team demonstrated that colicin A, which had been misidentified as colicin K (429), acts by making tiny pores in phospholipid bilayers, thus allowing the leakage of ions across them (575). The inner membrane (IM) was known to be the target of colicins that trigger the arrest of protein synthesis and active transport, since such colicins provoke various membrane perturbations (131a, 202, 203, 231a, 428a, 513a, 552).

The three steps of colicin action have thus been described. The colicin molecule causes killing after binding to a specific receptor on the outer membrane and being translocated through the cell envelope by either the Tol or TonB machinery to its target, which is the inner membrane for ionophoric colicins and the cytoplasm for nuclease colicins. Colicin M is a unique colicin acting on peptidoglycan synthesis through the enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors (176, 252).

No colicin acts on its own producing bacteria since each bacterium produces a specific inhibitor called the immunity protein. The immunity protein of pore-forming colicins is located in the inner membrane of producing cells (673), blocking colicin when it reaches its target after its entry into sensitive cells. In contrast, the immunity protein of nuclease colicins forms a complex with the cognate colicin in the producing cell, neutralizing its catalytic activity. It is this complex that is released. It dissociates only during colicin action on sensitive bacteria (35, 165a, 304, 590). The specificity of the interaction between the nuclease colicins and their immunity proteins has been extensively studied. The affinity of colicin E9 binding to Im9, its immunity protein, has been found to be in the femtomolar range; i.e., it is one of the strongest associations observed for a complex of two proteins (667).

In 1953, it was suggested that the ability to produce colicin resides in an extrachromosomal genetic element, named the colicinogenic factor, after the demonstration that the determinant for colicin is transmitted in mating experiments (122, 210). In 1965, De Witt and Helinski presented the first evidence that the colicin genetic determinants are located on plasmids (155). Further on, the various plasmids encoding the most studied colicins were isolated (20, 122, 356, 626). Two classes of colicinogenic plasmids, designated pCol, have been identified: small multicopy plasmids that contain the colicin operon and a mobilization factor and large monocopy plasmids that carry numerous genes besides the genes for colicin activity and are able to conjugate (251). Plasmid pColE1 was the first plasmid used as a cloning vehicle at the start of the era of genetic engineering in 1974 (263). Its genetic map was first drawn in 1978 (160), and its 6,646 bp were sequenced in 1985 (110). Since then, the complete sequences of pCloDF13 (9,957 bp) and pColA (6,720 bp) have been published (465, 480).

The organization of the colicin operon carried by the colicinogenic plasmids was first demonstrated in 1978 for colicin E1, an ionophoric colicin (160), and for cloacin DF13, a nuclease colicin (8). Both operons contain the SOS promoter followed by the structural gene for colicin. In the operon of enzyme colicins, the structural gene of the immunity protein is located downstream from the colicin gene and upstream from the gene encoding the lysis protein responsible for colicin release. The operon of pore-forming colicins does not contain the structural gene of the immunity protein: it is located on a specific operon under constitutive regulation present on the opposite DNA strand. The gene encoding the colicin lysis protein is always the last gene of the operon. It is present in the operons of group A colicins but not in those of group B colicins.

Colicins have been purified and found to be proteins of high molecular mass ranging from 40 to 80 kDa (131, 261, 262, 358, 579). Their amino acid sequences have been deduced from the nucleotide sequences of their structural genes; the first to be determined was colicin E1 in 1982 (681). None of the sequences contains disulfide bonds. All colicins are organized into three domains, each corresponding to one step of colicin action, as shown first by de Graaf et al. in 1978 and then by Ohno-Iwashita and Imahori in 1980 (140, 495, 496). The N-terminal domain is involved in translocation through the membrane, and the central domain is involved in binding to the receptor, while the C-terminal domain contains the active part. The pore-forming colicins are monomeric, whereas the enzyme colicins are heterodimers of colicin and the immunity protein.

Information on the structures of colicins has slowly emerged. They are elongated proteins (358) and are hard to crystallize. The crystal structure of the C-terminal domain of colicin A was obtained in 1989 (502), long before that of an entire colicin was obtained, which did not appear until 1997, when the structure of colicin Ia was solved (675). This has now been joined by several other colicin structures (177, 267, 607, 650). In most cases, the domain structure deduced from experiment is well explained by the crystal structure.

This report on the landmarks of colicin research during more than 80 years demonstrates that colicins have yielded numerous results pertinent to a variety of fields. The main data obtained on their mechanism of production and release, on the three steps of their mode of killing, on the specificity of the immunity towards them, and on the possible route of their evolution are considered below. Many of them have been reported in previous reviews (258a, 269a, 308, 356a, 552a). It was of interest to collect and compare them and to look forward to where colicins may lead us in the future.

COLICIN SYNTHESIS

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Various plasmids may encode a similar colicin. The best known case is that of colicin E1, which is encoded by type I plasmids as different as pML30 and pJC411. The sequence and organization of both plasmids and the amino acid compositions of their gene products are different, although the encoded colicin has the same characteristics: it is a pore-forming protein using BtuB as a receptor and the Tol system as a transit machinery. Other plasmids producing colicin E1 are known (D. Cavard, unpublished results). A nomenclature should be established to designate them using either the name of the plasmid, as colicin E1-ML30, or a letter, as colicin E1a. This case is not unique. Different plasmids encode colicins A, E2, E3, B, and D (561).

One case of a chromosomally encoded colicin has been reported: colicin-like bacteriocin 28b produced by Serratia marcescens, a colicin very homologous to pore-forming colicins. In this case, the structural gene of colicin is not associated with immunity and release genes in contrast to what was observed with plasmid-encoded colicins (239).

Col plasmid replication has been thought to be coregulated with colicin production since phage induction has often been compared to colicin induction and involves the replication of the phage genome (4, 155). That has been ruled out, since colicin biosynthesis can be induced when plasmid synthesis is inhibited (170, 289, 353).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Organization of the colicin operons. The genes are represented by arrowheads. SOS promoters (PSOS), the immunity promoter (Pim), and transcription terminators (T) are indicated by arrows. Names of the colicin gene (cxa, in which x is specific to the colicin) and its immunity gene (cxi) and lysis protein gene (cxl) follow the nomenclature.

The last gene of colicin operons is the gene encoding the lysis protein, named cxl for colicin X lysis protein, whose product allows the release of colicin into the medium and is responsible of the cell death after induction (colicins A [99], E1 [568, 660], E2 [123], K [515], N [535], U [599], and Y [558] and cloacin [244, 245]). It is present in the operons of group A colicins and in some operons of group B colicins such as those of colicins 5 (515), 10 (516), and D (268, 534, 564).

The colicin operons thus contain one to three genes. However, redundancies have been found in the organizations of many colicin operons. For instance, the colicin E3 operon contains two different immunity genes, one to colicin E3 and one to colicin E8 (105, 445), as does that of colicin E6, which contains the immunity genes to E6 and to E8 (2). The colicin E9 operon contains the immunity genes to colicin E9 and to colicin E5 and two lysis genes, that to colicin E9 and that to colicin E5 (107, 129, 383). A common origin of all colicin operons and an evolutional relationship of the colicinogenic plasmids have been suggested. The various colicins seem to have been assembled from a few DNA fragments that encode the functional domains of the proteins (2, 107, 129, 383, 446, 555, 564, 627, 671).

The organization of the colicin Js operon differs from that reported above. The cjl gene encoding the lysis protein is located upstream from the gene for colicin activity, cja (600). Colicin Js is a 95-amino-acid polypeptide of 10.4 kDa with no sequence similarity to other known colicins. It resembles microcins, although the pColJs plasmid that encodes it shows striking similarities with pColE1, and cannot be classified among colicins as previously reported (601).

Transcription of the colicin operons is strongly repressed by the LexA protein, the repressor of the SOS genes (reviewed in reference 409). Except for cloacin DF13, colicin operons contain two LexA boxes in tandem but overlapping by one or two bases (106, 123, 171, 411, 433, 445, 466, 537, 578, 599, 638, 649). The two LexA boxes have been found in every colicin operon studied so far and in the same organization and location. They are located just downstream from the Pribnow box. Each box binds one dimer of LexA. The fixation of two dimers provokes DNA bending, which adds to the blockade of the operon transcription (414). After DNA damage by mutagenic and carcinogenic agents, RecA is activated and stimulates LexA autocleavage and release from the LexA boxes, allowing transcription of the colicin operon. The agents that are able to trigger the SOS response and to induce colicin production are numerous and of different natures: from physical agents such as UV light to chemical drugs and stress conditions (430). The most popular agent used to induce colicin production in research laboratories is the antibiotic mitomycin C (286). Such DNA damage regulation is found for colicins and every class of protein bacteriocins but neither for peptide bacteriocins nor for microcins (128, 262).

Although LexA is the common repressor of colicin transcription, other repressors or activators play a role to modulate the expression of some colicin operons. For instance, transcription of the colicin E1 operon is stimulated by catabolite repression (172). A site of the cyclic AMP receptor protein-cyclic AMP complex has been identified on the promoter of the colicin E1 operon (171, 172), and a potential site for cloacin DF13 (638), colicin Ib (649), and colicin B (578) promoters has been found. Thus, regulators other than SOS agents interfere with colicin transcription. That and DNA bending may explain the pronounced lag observed in colicin expression after SOS induction (262), compared with the expression of other SOS genes (569). The lag is more or less significant according to growth conditions and to the bacterial strain used. Colicin synthesis has been shown to be stimulated in various cases by thymine starvation (589), stringent response (419), catabolite repression (172, 535), ompR mutation (543), the stationary phase of growth (185, 569), anaerobiosis (184), high temperatures (85, 86, 333), or nutrient depletion (368). In contrast, it is significantly reduced by low temperatures and in pldA null mutants (85, 88). Thus, various global regulatory proteins and environmental signals influence colicin synthesis, and some specific gene products are required. The expression of the colicin A operon is activated by both of its gene products: colicin A and Cal, the colicin A lysis protein (86, 87). A role of the E8 lysis protein in the regulation of colicin E8 synthesis has been reported (386). An activator has been found to be required for the transcription of colicin-like bacteriocin 28b (201). Regulation of the colicin operons is complex and may vary from one operon to the other. It looks like that of the virulence genes of various pathogenic bacteria, which depends on growth conditions (455), indicating that colicins, like virulence factors, play a competitive role in the wild environment. However, colicins are primarily under SOS control, and the reason for this is not yet clear.

Transcription from the SOS promoter of the colicin operons of group A results in the formation of two mRNA transcripts due to the presence of two terminators of transcription (Fig. 1). The major mRNA corresponds to the colicin gene for the operons of pore-forming colicins and to the colicin and the immunity genes for the enzyme-colicin operons. In this case, both colicin and immunity genes are coordinately transcribed and translated, and both genes products associate immediately after synthesis to form a dimeric complex devoid of enzymatic activity. In order to block nuclease colicins, an additional promoter is present upstream of the immunity gene (Fig. 1), allowing a higher production of the immunity protein than that of the colicin (105, 445). The minor mRNA is the largest one, as it corresponds to a transcript of the entire operon that is of both the colicin and the lysis genes for the pore-forming colicin operons and of the colicin, the immunity protein, and the lysis protein genes for the nuclease colicin operons (106, 411, 412, 637). Thus, the lysis gene is transcribed at lower levels than the colicin gene.

The translation of the mRNAs of colicins A, E2, and E3 is discontinuous. Discrete elongation intermediates are observed during colicin synthesis. That appears to be due to the tRNA availability for the various codons and to the presence of a high proportion of codons corresponding to rare tRNA in colicin genes (648). The presence of several rare codons in the colicin K mRNA allows ppGpp to regulate colicin K synthesis via a variable cognate tRNA availability (367). An autoregulation of the translational expression of the colicin E7 operon by the immunity protein to colicin E7 has been suggested (283).

Whether colicin is produced in small amounts by all the cells of a culture or in large amounts by a fraction of cells during either the spontaneous or the induced production of colicin has long been a subject of controversy. In 1959, Ozeki et al. tried to determine the number of individual cells producing colicin E2 at a given time by measuring the number of lacunae on a lawn of sensitive cells on a petri dish (501). One lacuna is due to the growth inhibition provoked by the amount of colicin produced by a single colicinogenic cell. They concluded that 0.1% of cells produce colicin E2 under normal conditions compared to 50% after UV irradiation (501). Recent work with colicin K labeled with green fluorescent protein (GFP) demonstrated that only 3% of cells produce colicin upon induction by nutrient starvation (471). That does not seem to be the case upon induction of a group A colicin by an SOS agent such as mitomycin C, which is always accompanied by the death of the total population of the colicinogenic culture (80a, 92, 93, 94, 262, 297). The death is not due to the produced colicin, as colicinogenic cells are protected against it by the immunity protein, but rather is due to the production of the lysis protein coexpressed with colicin.

Colicin operons of group B colicins contain a lysis gene when carried by plasmids of type I, as colicins 5 and 10, but do not contain a lysis gene when present on type II plasmids, except that of colicin D (268). Thus, their synthesis is not lethal for the producing cells and is not followed by the release of colicin into the extracellular medium.

COLICIN RELEASE

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As first observed by Gratia, colicins are present in the culture medium of producing bacteria (235), and their release into the environment has long since been thought to be a unique form of protein secretion by E. coli. Colicin release involves only one gene product, the colicin lysis protein, also referred to as the killing (kil) protein or bacteriocin release protein (BRP) (245, 303, 500, 541, 568, 641), and takes place following the synthesis of both colicin and the lysis protein. Lysis proteins allow colicins to be released into the medium. The mechanism of colicin secretion differs from that of the five secretion pathways in gram-negative bacteria known to be involved in the release of proteins into the extracellular medium (reviewed in references 481 and 623). It also differs from the mechanism of action of phage lysis proteins that provoke the total lysis of phage-producing bacteria (reviewed in reference 690). It does not involve autolytic enzymes, which hydrolyze the peptidoglycan, as do many phage lysis proteins (279), nor is it related to the secretion system of microcins such as colicin V, which is mediated by an exporter system consisting of two specific cytoplasmic proteins, CvaA and CvaB, and of the host outer membrane protein TolC. CvaB is a member of the ATP binding cassette (ABC) superfamily. The amino-terminal export signal of colicin V, which is a double-glycine leader sequence specific for the CvaA-CvaB-TolC exporter, is processed concomitant with secretion (226, 242). The export of colicin V instead resembles that of various peptide bacteriocins produced by gram-positive bacteria, which also require the activity of a dedicated ABC transporter. The transporter is the maturation protease that cleaves off the typical double-glycine leader sequence of the peptide bacteriocins concomitant with their translocation across the membrane (162, 254, 294).

The gene encoding the lysis protein is the last gene of the operons of group A colicins. Release does not occur in cells missing the gene, such as bacteria producing a group B colicin, nor does it occur in bacteria containing mutations, insertions, or deletions in the lysis gene (99, 244, 245, 303, 541, 568, 627). Transcription of the lysis gene relies upon transcriptional readthrough from the promoter of the colicin structural gene across the entire operon. Thus, the colicin lysis protein is coexpressed with colicin; conversely, colicin might be synthesized without the lysis protein due to a transcription terminator at the end of the gene (Fig. 1). The lysis protein is always produced in lower amounts than colicin (106, 411, 412, 637) but in significant amounts (similar to that of Lpp, the murein lipoprotein). Induction of the lysis gene cloned under various promoters provokes protein release and death of the host, even in noncolicinogenic cells (3, 94, 423, 541).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Sequence alignment of colicin lysis proteins. Amino acid sequences of the colicin lysis proteins are shown. The sequences of the colicin lysis proteins encoded by the colicinogenic plasmids indicated at the left are presented. Identical amino acids are in boldface type. The numbers of residues in the signal peptide and in the mature form are indicated. X63621, EMBL/GenBank/DDBJ accession number X63621.

Lysis proteins are acylated and processed in many steps, as are all bacterial lipoproteins. They are synthesized in the cytoplasm as precursor forms, which are driven by the signal sequence to the inner membrane and translocated by the Sec machinery to the outer leaflet of the inner membrane, where they are modified by Lgt, the lipoprotein glyceryl-transferase, with a diglyceride on the sulfydryl group of the cysteine residue of the lipobox. Next, the modified precursor forms are processed by LspA, the signal peptidase II specific for lipoproteins, into apolipoprotein and signal peptide. The amino group of the acylated cysteine is then acylated by Lnt, the lipoprotein N-acyltransferase (679, 689).

The rate of acylation and processing varies from one lysis protein to another; this is rapid for CelA, the colicin E1 lysis protein, but of the order of several minutes for Cal, the colicin A lysis protein, and BRP, the cloacin DF13 lysis protein. Consequently, three forms of Cal and BRP are present following induction: the precursor, the modified precursor, and the mature forms. The kinetics of Cal maturation do not vary regardless of whether the lysis protein is coexpressed with colicin or alone. However, the levels of the three forms of Cal vary with time and depend on the genotype of the host strain (81, 84). The signal peptides of Cal, CelB (colicin E2 lysis protein), and BRP are stable after cleavage and accumulate in the inner membrane, while that of CelA is immediately degraded (80a, 84, 91, 99, 424, 425, 539). The dependence on the Sec machinery also varies from one lysis protein to another: CelA and BRP are Sec dependent, while Cal is Sec independent (81, 499).

The final location of lipoproteins depends on the residue at position 2 (218, 683). They are anchored to the periplasmic leaflet of either the inner or outer membrane through fatty acyl chains covalently linked to the N-terminal cysteine. The inner membrane retention signal for lipoproteins has Asp at position 2 in combination with certain residues at position 3, which functions as a Lol avoidance signal, since this inhibits the recognition of lipoproteins by the five proteins of the Lol system, which release lipoproteins from the inner membrane (622). All colicin lysis proteins possess a glutamine residue at position 2 and so should be recognized by the Lol system and located in the outer membrane. The mature forms of cloacin DF13 and colicin A, E2, and E3 lysis proteins have been observed in both the inner and outer membranes, while that of colicin N has been found only in the outer membrane (123, 277, 303, 500, 538). The presence of lysis proteins in both membranes is due to their slow assembly in the outer membrane. Maturation and assembly have been measured for Cal by sucrose gradient analysis of radiolabeled membranes of colicin A-producing cells at various times of induction (277). Both Cal and CelA take the same time to acquire the specific electrophoretic behavior of outer membrane proteins; both lysis proteins are soluble in SDS after maturation, but, many minutes later, they have to be heated in SDS to be solubilized at the time of colicin release. They are then partly released into the extracellular medium with colicin. The exported lysis proteins share the electrophoretic property of outer membrane proteins, indicating that they are kept in a structure similar to that before export (80a, 81). No colicin lysis protein has been purified to homogeneity thus far, which has limited their biochemical characterization. In addition, lysis proteins are not readily soluble and are prone to aggregation.

Functions of Colicin Lysis Proteins The main function of the lysis protein is to promote colicin release, and so they are more correctly termed colicin release proteins, Cxr (for colicin X release). Concomitantly, they provoke quasilysis; modifications of the structure of the cell envelope; activation of OmpLA, the outer membrane phospholipase A; and death of the producing cell. However, the chronology of the various events is not well understood and seems to appear simultaneously. The functioning of lysis proteins does not depend on colicin production or on RecA-LexA regulation. It does, however, require that the lysis protein be produced in reasonable amounts. The various physiological changes provoked by lysis proteins occur late after synthesis, indicating that a critical concentration of the lysis protein in the host cell is needed. There is no specificity associated with lysis functions since colicin lysis proteins are similar (Fig. 2) and therefore interchangeable (541).

Colicin release. After induction by DNA-damaging agents, colicins are expressed and accumulate in the cytoplasm of the producing cells (96). Group A colicins that are synthesized with a mutated or a deleted lysis protein and colicins of group B (which are not coexpressed with a lysis protein) remain in the cytoplasm during and after induction. Colicins do not possess signal export domains; they have neither cleavable N-terminal signal sequences, which can mediate their transport to the periplasm, nor internal export domains, which could help them to be secreted by the lysis protein (17). Some mutations of colicins A, E1, and E3 and cloacin DF13 that inhibit release have been identified (7, 15, 461, 646, 682), but this could be due to aggregation or to changes in conformation, as these mutated colicins were inactive. On the other hand, mutations of the colicin lysis proteins have been reported to block release, as described below.

After induction, colicins A and E2 and cloacin DF13 are found in both the cytoplasm and periplasm (88, 96, 500, 540). Part of colicin A has been shown to be localized in the outer membrane, associated with porins and other outer membrane proteins (88). Colicins are progressively released into the extracellular medium 60 to 90 min after induction. Some periplasmic and cytoplasmic proteins are found in the medium with colicin (17, 99, 303, 461, 540, 541, 613, 647), but it is not known whether these proteins come from some lysed cells or through the lack of specificity of colicin release. They are found similarly after induction of a cloned lysis gene.

The level and timing of colicin release vary with growth conditions. Release is slowed down in the presence of divalent cations (20 mM), as was seen for colicins A, E1, and E2 and cloacin DF13 (3, 93, 426, 542). It is also significantly retarded in colicin A-producing bacteria induced at low temperatures. In contrast, colicin A release is sped up at elevated temperatures, after a heat shock, in the presence of either EDTA or Triton X-100, or in hosts carrying a mutation in the degP gene (85, 93, 98). Such variations seem to be due to the cellular concentration of the lysis protein, since the amount of Cal is higher in the presence than in the absence of EDTA and is lower in the presence than in the absence of Mg²⁺ ions (84). The level of the colicin lysis protein is modulated by the same environmental factors as colicin and is directly responsible for the rate of colicin release. The cellular concentration of the lysis protein might also be regulated by proteolysis, as some stress conditions provoke the induction of proteases. In summary, the amount of lysis protein appears to be critical for its function in colicin release from bacteria.

Quasilysis. The optical density of a culture of bacteria producing a group A colicin increases for about 60 min after induction, as does that of a noninduced culture. It then decreases significantly, reaching an optical density similar to that at the time of induction (297). This drop of absorbance was called quasilysis by Jakes and Model in 1979 (300) and occurs whether or not colicin is produced (94, 423). It does not correspond to complete lysis as seen after the induction of lysogenic bacteria, as no lysed bacteria or membrane fragments have been observed during this period (92).

Quasilysis is a good reporter of the functioning of colicin lysis proteins. Both quasilysis and colicin release take place simultaneously and vary according to growth conditions as described above. Neither occurs in pldA cells (542). In colicin A-producing cells, both are reduced in an rpoH mutant but increased in a degP mutant (85, 98). However, colicin A release occurs without quasilysis in cells incubated in the presence of 20 mM divalent cations and in cells grown at low temperatures. In both cases, the synthesis of colicin A and Cal is significantly retarded, allowing the growth of cells, which may mask the decrease of culture turbidity normally linked to colicin A export (85, 93).

Modifications of cell envelope structure. After colicin induction, changes in the structure of the cell envelope occur. Analysis on sucrose density gradients of cell envelopes for cells overproducing the colicin A lysis protein showed that both inner and outer membranes cannot be separated during the latter stages of induction (277).

Activation of OmpLA, the outer membrane phospholipase A. Neither colicin release nor quasilysis occurs in bacteria containing a mutated pldA gene (542). OmpLA, the pldA gene product (60), is an inactive monomeric protein which dimerizes when activated (141, 603). OmpLA is activated after the induction of colicins A and E2 and of cloacin DF13 (91, 426, 542). Its activation is due to the production of the lysis protein, as demonstrated by OmpLA dimerization after the induction of a subcloned BRP, the cloacin DF13 lysis protein (142). OmpLA activation has been postulated to cause colicin release, quasilysis, and killing of host cells. It provokes the formation of lysophospholipids, which are detergents and would permeabilize the outer membrane and, subsequently, the inner membrane of the cells (542). However, both colicin A release and quasilysis occur in the absence of an active OmpLA in a tolQ mutant (277). Functioning of lysis proteins is decreased in the presence of divalent cations, and that of Cal is increased in the presence of EDTA, despite the fact that OmpLA requires Ca²⁺ ions to be active (60, 141). OmpLA activation seems to be a consequence of the mechanism of lysis protein action and might be the cause of death of producing cells, as various lethal agents are known to provoke OmpLA activation (101).

The presence of an inactive OmpLA in the cell induces the Cpx and ^E regulons and consequently switches on many genes including genes encoding proteases such as degP (375). The amount of Cal is significantly reduced in cells with a missense mutation in the pldA gene compared to that of wild-type (WT) cells, suggesting proteolysis. The introduction of a degP mutation in pldA cells does not restore the level of Cal, indicating the presence of a protease(s) other than DegP that might be able to degrade the various Cal forms in cells with an inactive OmpLA (84). The induction or activation of proteases by a pldA mutation might explain why cells containing a null pldA mutation produce only traces of colicin A, in contrast to cells containing an inactive OmpLA (88). The induction of a protease(s) would be less significant in the missense pldA mutant than in the null pldA mutant in which both colicin A and Cal might be degraded by proteases.

Death of the host cell. The production of lysis protein, induced via the SOS or lac promoter, causes the death of the host cell whether or not colicin is expressed (3, 93, 98, 244, 245, 423, 541). Cell death is thought to be caused by OmpLA activation (542), which is a lethal event in bacteria (101, 141). However, the colony-forming ability of colicin A-producing cells that possess an inactive OmpLA decreases after induction although less significantly than that of wild-type cells. The survival of bacteria is altered by various treatments that affect quasilysis. Heat shock and the addition of EDTA or Triton X-100, for example, increase both the lethality and quasilysis of colicin A-producing cells, while divalent cations and low temperatures decrease them (85, 93). Surviving bacteria seem to be uninduced cells, cells in which colicin induction is retarded, or cells in which the two gene products of the colicin operon have been proteolysed. These cells multiply during induction, and their progeny may mask the death of the induced cells and quasilysis.

Mutants of E. coli that are resistant to both the lethal and lytic action of celA (kil) gene expression have been isolated. Some mutants are unable to produce colicin, while others are leaky to periplasmic proteins. The latter type of mutant releases colicin without OmpLA activation, but none of them die after induction. This suggests that CelA, the colicin E1 lysis protein, acts in various steps, each one involving various cell components (3, 614).

Regulator of expression of the colicin operon. Lysis proteins appear to play a role in the expression of the related colicin and in their own expression. The presence of a transposon in the lys gene of colicin E8 has been shown to reduce colicin E8 synthesis significantly, suggesting a role for the E8 lysis protein in the regulation of colicin E8 synthesis (386). A similar role for Cal (the colicin A lysis protein) as an activator of the transcription of the colicin A operon has been reported, as colicin A synthesis does not occur in the presence of globomycin in cells with a nonfunctional cal gene (86, 87).

Structure-Function Relationships of Colicin Lysis Proteins A surprising feature of colicin lysis protein biology is that although the proteins share a high degree of sequence conservation, mutagenesis experiments have shown in fact that only acylation, processing, and length of the lipopeptide are required for function. Acylation of the cysteine residue of the lipobox is essential for function. Changing the lipobox cysteine inactivates the protein. However, some colicin release has been observed when the cysteine of the colicin E2 lysis protein is replaced by a glutamine (539). The colony-forming ability is inhibited when the cysteine of BRP is replaced by glycine (425). Mutated Cal lysis proteins, which contain either proline or threonine in place of the cysteine residue in position 1, are rapidly hydrolyzed (91). The enzyme responsible for the degradation of the mutated precursor forms of Cal has been shown to be DegP, a periplasmic heat shock protein that combines refolding and proteolytic activities (363). In a degP null mutant, the precursor forms of C1P and C1T Cal remain stable and unprocessed in the inner membrane but nonfunctional (90).

Processing is absolutely required for function. Globomycin, an inhibitor of LspA, the signal peptidase of the lipoproteins (288), blocks the processing of the modified precursor form of lysis proteins and inhibits colicin release. In the presence of globomycin, the modified precursor form of CelA is rapidly hydrolyzed, while that of Cal is cleaved into two acylated fragments in wild-type cells. It accumulates uncleaved in a degP mutant, indicating that DegP hydrolyzes the modified precursor form of Cal, pCal^m (98), as the unmodified Cal precursor, pCal (90). The two sites of DegP cleavage on Cal seem to be located between Val14-Ser15 and Val23-Ser24, upstream of Met25, in agreement with reported DegP sites (355). They are therefore present in the three forms of Cal, pCal, pCal^m, and Cal, all of which might be substrates for DegP. In the absence of globomycin, two truncated Cals are observed by immunoblotting, while they are not seen with radioactive methionine labeling (84, 98). These short Cals might be formed either by DegP cleavage of the Cal mature form or by acylation and processing of pCal and pCal^m fragments produced after DegP action.

Despite the presence of DegP in wild-type cells, the three forms of Cal always seem to be present, indicating that the majority of Cal avoids DegP degradation. Various hypotheses might explain such an avoidance mechanism. First, DegP sites on Cal may be masked, for example, due to the formation of protein complexes with chaperones and/or other proteins or to polymerization. Second, the maturation of Cal might take place in locations that do not contain DegP, although it is known to occur at the periplasmic side of the inner membrane, where DegP is located. Third, Cal is produced in large quantities that might overwhelm DegP, which is a relatively minor protein. Synthesis of DegP is induced after heat shock, during stress conditions (610), and upon the overproduction of some outer membrane lipoproteins (459). Membrane anchoring of the lipoprotein NlpE is essential for degP induction, since the nonlipidated derivative of NlpE does not induce it (604). DegP synthesis may be induced during globomycin treatment by the accumulation of pCal^m. Finally, DegP combines two activities and may be switched to chaperone activity during colicin induction and to protease in the presence of globomycin. It is known that the chaperone function dominates at low temperatures, while the proteolytic activity is present at elevated temperatures (610). However, the level of the three forms of Cal present in degP cells is higher and Cal functioning is more significant than in degP⁺ cells, indicating that a certain amount of either form of Cal cannot escape DegP cleavage (84).

DegP sites are not present in all lysis proteins (Fig. 2), explaining why DegP degradation is not observed for CelA, the colicin E1 lysis protein. The modified precursor form of CelA accumulates neither in wild-type nor in degP cells after globomycin treatment, indicating that it is the substrate of a protease(s) other than DegP (81).

The signal peptide of the lysis protein is either unstable, like that of CelA and all lipoproteins, or stable, as seen in Cal and BRP. In these latter cases, it accumulates in the inner membrane in contrast to other leader sequences, which are hydrolyzed by signal peptide peptidases. This stability might be the cause of lysis and death of the host cell. However, quasilysis and cell death occur in colicin A- and colicin E1-producing cells even though the Cal signal peptide is stable and that of CelA is unstable (81). Cloning of the signal peptides of the colicin E2 and cloacin DF13 lysis proteins causes quasilysis, lethality, and some colicin release (322, 642). The replacement of the stable BRP signal peptide by the unstable signal peptide of the murein lipoprotein Lpp inhibits cloacin DF13 release but provokes killing, quasilysis, and leakage of periplasmic proteins. The construction of these hybrid precursors indicates that the BRP signal peptide is responsible for the slow processing and contributes with mature BRP to the transfer of cloacin DF13 across the cell envelope (642, 643). In vitro mutagenesis of the Cal signal peptide demonstrates that the Ile residue at position 13 is important for stability and that both Ile13 and Ala18 contribute to the slow modification and processing of the Cal precursor. However, it does not influence colicin A release, quasilysis, and death of the cell host, which are caused only by the mature form of Cal (280).

Although the composition of the mature form of colicin lysis proteins is particularly well conserved, mutagenesis of Cal has shown that many residues/regions are unimportant for function; changes to the conserved residues of the N-terminal half of Cal are without effect, except when a negative charge is present, indicating that charge plays some role in its function (276). More important is the length of the mature lysis protein. Truncated colicin E2, colicin E3, and cloacin DF13 lysis proteins of 20 amino acids are active, while a shortened Cal of 18 residues and a BRP of 16 residues are not (276, 422, 627). The length of the mature form plays a role in the rate of processing and maturation. Truncated Cal containing the first 16 or 18 residues is neither acylated nor processed, except when overproduced, indicating that its maturation is slower than that of wild-type Cal, explaining its lack of activity (82). In contrast, amino acid extensions to lysis proteins do not seem to modify their functions. A hybrid protein of BRP-lactamase functions as BRP for the export of cloacin DF13 (424).

Colicin lysis proteins are homologous to VirB7, the lipoprotein of the type IV secretion system of Agrobacterium tumefaciens (90, 586). VirB7 is a 41-amino-acid lipoprotein that forms homodimers and heteromultimeric complexes with other putative outer membrane proteins of the VirB system (reviewed in reference 76).

Colicin lysis proteins share some similarities with the lysis proteins of single-stranded phages (384). The main difference between them is that the colicin lysis proteins are lipoproteins, while phage lysis proteins are not. However, a lipoprotein homologous to the colicin lysis protein, called Rz1, is encoded by a reading frame embedded within the Rz lysis gene of phage . It is localized in the outer membrane and plays a role in the timing of lysis in lysogenic cells following induction (328, 700). Similar lipopeptides have been shown to be encoded by the lysis genes of other bacteriophages such as P2 and N4, which form heteromeric complexes and are required for host lysis (436).

Mechanism of Action of Colicin Lysis Proteins How the colicin lysis protein allows colicin release has not been fully elucidated. The majority of secretion systems thus far described for transporting proteins across the membranes of gram-negative bacteria utilize multiprotein machines. Colicin release differs from all these secretion systems not only by the number of proteins required for transport but also by the amounts of both the protein transported and the protein transporter, the timing of the release, and the lack of specificity. Both colicins and their lysis proteins are produced in large amounts in the cell, with colicin in higher concentrations than the lysis protein. Most of the colicin is released into the medium, while only some of the lysis protein is released, and this occurs only late in the induction/release process. The lag before release is greater than a generation time, suggesting that modifications and/or the buildup of structures might occur.

Various models to explain the mode of action of lysis proteins have been proposed. For example, they could form pores through both the inner and outer membranes. The colicins located in the cytoplasm would then cross both membranes through these putative trans-envelope pores. The stable signal peptide present in the inner membrane might participate in the formation of such pores, which would destabilize the cell envelope and provoke OmpLA activation (139, 643).

Recent studies described two steps of colicin A release by Cal, the colicin A lysis protein (90). In the first step, colicin A produced in the cytoplasm moves to a location where it can be extracted by washing. This location is presumably the periplasm, in which colicin A and other colicins produced in the presence of a lysis protein have been detected. Cal is required for this step, suggesting that it helps colicin A cross the inner membrane. This step is slowed down in cells with a nonacylated Cal precursor, pCal, suggesting that both pCal and colicin A may be associated and translocated simultaneously to the periplasm. In the second step, colicin A is released into the medium with some Cal. During both steps, colicin A is found in various forms, as described previously (90). In particular, hetero-oligomers of colicin A bound to OmpF and OmpC porins, called colicin Au, are present during each step. Colicin A present in the periplasm would associate by its C-terminal domain with porins and also to other outer membrane proteins already incorporated into the outer membrane, constituting colicin Au, the formation of which is Sec independent. In vitro binding of the C-terminal part of colicin N with porins has been reported (161). It has been proposed that the colicin Au structure might be similar to that of TolC (88), i.e., a ß-barrel of porins fused to an -helical tunnel made of the C-terminal domain of colicin A (359). This structure would allow cellular material to go out and would be dangerous for the producing cells except in the presence of Cal. In the absence of Cal, colicin Au is unstable, and the cells stop synthesizing OmpF, indicating that OmpF is lethal for them. Cal seems to associate with colicin Au, as it is found in purified colicin Au fractions, and to stabilize and detoxify them. It is proposed that this multimeric complex constitutes part of the machinery for colicin export.

Colicin A present in the periplasm might also associate with the Tol proteins, which would become nonfunctional. Such an interaction in cells producing the N-terminal domain of colicins has been described (43). The producing cells exhibit phenotypes similar to those of tol mutants, which are leaky and produce outer membrane vesicles (28). In this instance, vesicles would contain Cal located in the outer membrane and colicin A present in the periplasm. The outer membrane vesicles would be released from bacteria, explaining the presence of both lysis proteins and colicin in the medium (90). Vesicle-mediated export for the release of toxins and for sending signals from cell to cell has been described (447, 657). However, the formation of such vesicles, as that of the colicin export machinery, requires periplasmic colicin and does not explain the mode of action of separately expressed lysis proteins.

Current evidence suggests that colicin release has similarities to colicin entry. As described above, colicins are able to interact with outer membrane proteins such as porins and the Tol machinery in the periplasm. Both the uptake and the release of colicin provoke OmpLA activation (91, 101). Such a