REVIEW Folia Microbiol. 43 (6), 563-582 (1998)

Colicins Exocellular Lethal Proteins of Escherichia coli* J. ~MARDAand D. ~MAJS Department of Biology, Faculty of Medicine, Masaryk University, 662 44 Brno, Czech Republic Received March 17, 1998

ABSTRACT. Colicins are toxic exoproteins produced by bacteria of colicinogenic strains of Escherichia coli and some related species of Enterobacteriaceae, during the growth of their cultures. They inhibit sensitive bacteria of the same family. About 35 % E. coli strains appearing in human intestinal tract are colicinogenic. Synthesis of colicins is coded by genes located on Col plasmids. Until now more than 34 types of colicins have been described, 21 of them in greater detail, v/z. colicins A, B, D, El-E9, Ia, Ib, JS, K, M, N, U, 5, 10. In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell. The classificatiQn of colicins is based on differences in the molecular events of these three steps.

CONTENTS 1 2 3 4

5

6 7 8 9 10

1

Brief characterization of colicins 563 Classification and nomenclature of colicins 564 Occurrence of colicinogeny 564 Production of colieins by colicinogenic bacteria 565 4.1 Col plasmids 565 4.2 Molecular organization of colicin synthesis 568 4.3 Export of colicins from the producer bacteria 569 Interaction of colicins with bacterial cells 570 5.1 Colicin-receptor binding 571 5.2 Colicin translocation through the cell envelope 572 5.3 Lethal effect ofcolicins 573 5.3.1 Colicins depolarizing the plasma membrane 573 5.3.2 Nuclease colicins 574 5.3.3 Colicin M and pesticin I 574 Immunity to colicins 574 Inhibitory effects of colicins on bacteria deprived of the cell wall, and on eukaryotic cells Evolution of colicins and of Col plasmids 575 Practical applications of colieinogeny and colicins 578 Conclusions 578 References 579

BRIEF CHARACTERIZATION

575

OF COLICINS

Colicins are toxic exoproteins, produced by bacteria of colicinogenic strains of Escherichia coli and of some related species of the family Enterobacteriaceae during the growth of their cultures (Gratia 1925; Gratia and Fredericq 1946). They exert an inhibitory effect on sensitive bacteria of the same family. The effect is usually mediated by specific receptors in the cell wall. They are thus members of the large group of bacteriocins. It is typical of the bacteriocins of Gram-negative bacteria that both the producer and the sensitive strains belong to the same family and, in most cases, to the same species (Pugsley 1984a,b). However, not all bacteriocins of the family Enterobacteriaceae are designated as colieins, even though some of them have the same basic features, and are related to them in their primary structure. While bacteriocins of the species Shigella and Serratia have been considered as colicins since the beginning of the classification of bacteriocins, bacteriocins of the genera Enterobacter, Yersbda, and Serratia have been given different names, viz. cloacins in Enterobacter cloacae, pesticins in Yersinia pestis, and marcescins in Seratia marcenses. In this paper we shall maintain the conventional terminology, although

*The original version of this review was published in Czech in the journal "BiologiclMlisty" 62, 107-130 (1997).

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we shall consider, in addition to colicins, some related, well-defined, bacteriocins of the family Enterobacteriaceae. Colicins are a heterogeneous group of proteins with antimicrobial activity. In contrast to microcins (Baquero and Moreno 1984) they are proteins or small polypeptides, rather than oligopeptides only. Colicins are "SOS"-inducible, e.g., by mitomycin C, or by UV-radiation. They are not modified posttranslationally (with the possible exception of the removal of methionine from the N-terminus). They differ from microcins also in the type of export from the producer cells. Their differences from low-molar-mass antibiotics include, beside the protein structure and other parameters, the fact, that colicin synthesis reaches its maximum usually during the exponential phase of growth of the producer culture. Although research into colicins started and continued for a long time in parallel with the bacteriophage research (Gratia 1932; Jacob et al. 1952) and although some bacteriocins (from the family Enterobacteriaceae) have a corpuscular character (~marda 1987), no sequence homology of colicins with bacteriophage proteins exists. Hence, colicins and colicinogeny (i.e. the ability of bacterial strains to synthesize them) are independent phenomena of molecular microbiology. The first bacteriocin discovered was colicin V (Gratia 1925), originally described as "principle V", and classified as microcin V today. At present, 34 types of colicins have been differentiated, 21 of which are known in detail (colicins A, B, D, E l - E 9 , Ia, Ib, .Is, K, M, N, U, 5, 10; see Table I).

2

CLASSIFICATION A N D N O M E N C L A T U R E OF COLICINS

Classification and nomenclature of colicins are closely related to their principal mode of interaction with sensitive bacterial cells. This interaction proceeds in three steps: (/) binding of colicin to a specific receptor of the outer bacterial membrane, (ii) translocation of colicin through the cell envelope, and (iii) lethal interaction of colicin with the specific intracellular target (Reeves 1965). According to the original scheme, colicins are classified and designated on the basis of their receptor specificity (or of the absence of cross-resistance of the receptor mutants) by capital letters (Fredericq 1946, 1948). If more types of colicins are bound to the same receptor, they are further distinguished on the basis of absence of cross-immunity of the producer strains (Fredericq 1965), and marked by means of indices added to the type letter (usually by numbers, sometimes by letters). The strains producing the same colicin produce also the same immunity protein (see part 4.2). There exist, however, many exceptions from this general rule: colicins B and D bind to the same receptor in the outer bacterial membrane FepA (Hantke and Braun 1975), colicins G and H to the Fiu receptor (Bradley 1991), colicins Ia, Ib, and $1 to the Cir receptor (Pugsley 1984a; Ferber et al. 1981), etc. Besides, some colicins are designated -- without any effort to adhere to the general rules -by numbers or codes of the producing bacterial strains considered as their natural producers (Bradley and Howard 1992; Viejo et al. 1992). Colicins X and V have been reclassified as microcins (Baquero and Moreno 1984; San Millfin et al. 1987), as soon as this category of substances had been established (Baquero et al. 1978). This classification is not only difficult to employ (due to the many exceptions), but it is also not complete. An important aspect of the colicin classification is also the type of translocation mechanism, which is used by colicins for the transport through the cell envelope. Group A make use of some of the proteins TolA-, B-, C-, Q-, R- (the Tol system); group B make use of proteins TonB, ExbB,D (the Ton system) (Davies and Reeves 1975a,b). For example, colicins K and 5 are recognized by the Tsx receptor, and producers of both are cross-immune to each other; however, they are distinct colicins, since they are transported through the cell envelope by different translocation systems (Pilsl and Braun 1995c). It is therefore important for an exact classification of the colicin to know its receptor specificity (or the cross-resistance of the receptor mutant, selected by it, with other colicins), the type of translocation mechanism used by it, and the presence or absence of the cross-immunity of its producer toward the colicins which use the same receptor.

3

OCCURRENCE OF COLICINOGENY

The colicinogenic strains, i.e. strains synthesizing one or more types of colicins, form a substantial part of natural (and clinical) isolates of E. coli strains. The literature data concerning the

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occurrence of colicinogeny, show a wide variety, and can be considered as significant to different degrees, with respect not only to different samples tested and test methods applied, but also partly to the different indicator strains used by individual authors. Moreover, some studies wrongly interpret any growth inhibition recorded as being due to colicins. The data on frequency of colicinogenie strains in the species E. coli usually fall into the interval of 25-45 % (Brandis and gmarda 1971). Values not comprised in this interval, such as 12 % (Gardner 1950), or 75 % (Fredericq et al. 1949), are to be considered as exceptional. The frequency of colicinogenic isolates is evidently influenced by the clinical conditions in the host's bowel. Thirty percent of the lactose-positive strains of Gram-negative rods isolated from the river Seine produce one or more colicins, affecting the indicator strain E. coli K12 (Pugsley 1984b). The reference collection of E. coli strains ECOR keeps 35 % colicinogenic ones, where samples originated from human sources show a higher incidence. The high incidence of colicinogeny in natural isolates, particularly in isolates from the human large intestine, together with the finding that colicins are active even in the large intestine content (J. Tr~ka, personal communication), support a significant ecological importance of this phenomenon. Colicinogenic bacteria have an apparent selective advantage over the non-colicinogenic ones: they kill competitive bacteria of other strains of the same species, while themselves being specifically immune to the colicin they produce (though not to others). Cells of E. coli represent only 0 . 1 - 1 % of the total bacterial population in the human large intestine (Sonnenborn and Greinwald 1~)1) but they may have an important function for the interaction between the human organism and its intestinal flora, occupying an ecological niche on the surface of the intestinal mucosa epithelium. E. coli utilize oxygen diffusing through the mucosa into the intestinal lumen, creating suitable conditions for the growth of strict anaerobes, which represent most of the intestinal bacteria. In this situation, the ability of colicinogeny may be an important factor, in favor of representatives ofE. coli against other species of the Enterobacteriaceae family adapted to the same environmental conditions.

4

P R O D U C T I O N OF COLICINS BY COLICINOGENIC B A C T E R I A

4.1 Col plasmids The synthesis of colicins is coded by genes on the so-called Col plasmids (Fredericq and BetzBareau 1953; Clowes 1963) which vary widely in size. For example, pColE1 measures 6.6 kbp, while pColH is as much as 94 kbp long (Chan et al. 1985; Bradley 1991). According to the size, ability of amplification, number of copies per cell and ability of independent transfer by conjugation, the Col plasmids can be divided into three groups (Pugsley 1984a): (1) Group Ia: small plasmids (3-6 MDa), appearing in marly copies (15-30 spontaneous copies, without induction) in a cell, able to replicate even without protein synthesis by the host cell (amplification), but unable of an independent transfer by conjugation (Tra-); (2) Group/b: small plasmids, appearing in many copies, unable of amplification, or of transfer by conjugation ( Tra-); (3) Group Ih large plasmids (70-90 MDa), appearing in a few copies, unable to amplify, but often capable of transfer by conjugation (Tra § Groups Ia and/b of Col plasmids have been better described so far than the group H. In addition to structural genes, coding for the synthesis of colicin sys'~em proteins (of colicin, immunity protein, and lysis protein, see part 4.2), Col plasmids can code also for further functions. Mainly for this reason, it is advisable to add the name of its original host strain for an exact identification of a Col plasmid, for instance Col E l - K 3 0 (Reeves 1972). The group Ia of Col plasmids is represented by pColE1 (Fig. 1), coding for the synthesis of colicin El; it is a completely sequenced plasmid with 6646 base pairs, counted from the only EcoRI cleavage site. The plasmid pColE1 bears three genes of the colicin operon (see part 4.2), and seven for the functional regions (Chan et aL 1985). As an example of the group/b, pColG may be quoted. The rep region directs replication. The site for joining the first deoxyribonucleotide of the daughter plasmid designated as the start (origin) of replication (ori), is in position 1196. A part of the rep region is also the region inc, which is responsible for the incompatibility of the plasmid and for the control of its copy number, and the born region around the nic site 1468, binding the Mob protein (or proteins). During conjugation initiated by the sex factor F, the DNA chain with Mob proteins is transported through the conjugation pore into the recipient cell. The rom region codes for a small protein

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J. ~ M A R D A

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(63 amino acid residues), which suppresses the pro~iii.:.:. duction of the primer and decreases the number of plasmid copies in the cell. This type of regulation is cea ~ ~ . essential for amplification, i.e. for the continuing repli..::::::" I/1C cation of the plasmid even after blocking protein synthesis in the bacterial cell by chloramphenicol, which .:ii:: inhibits replication of chromosomal DNA. The inhibition of the Rom protein synthesis switches off this negative regulation of the plasmid replication initiation, and the surplus of some not used replication proteins makes it possible to increase the number of its copies in the cell to several thousand. Proteins of the m o b region serve to mobilize a plasmid by another, conjugative, plasmid. The cer region is necessary for a RecA-, RecE-, and RecF-independent recombicot nation, and for the conversion of plasmid multimers to Fig. L Map of the colicin plasmid E l (pCoIE1; 6 646 bp). Positions of specific regions are given in monomers. The exc region codes for proteins which relation to the unique restriction site for the decrease the efficiency of the DNA conjugation transendonuclease Abbreviations are explained in fer in the pairs of conjugating cells carrying a homothe text. logous pColE1. The group H of plasmids carries -- beside the colicin system -- a number of other functional regions coding, for instance, for the ability to conjugate, factors of adherence to tissues, the aerobactin-mediated uptake of iron from the environment, serum resistance, etc.

/

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Sr ~ nI~Om

EcoRI.

(i.e.

The colicin genetic system (colicin operon) of Col plasmids gene arrangement and orientation, regulation of their expression, is similar in all the three groups, and creates a relatively autonomous system in them. It can also indirectly serve to stabilize a given plasmid in a strain: cells that have lost their plasmid (which happens regularly, although with a low probability) can be eliminated from its population by colicin from other cells, as long as they carry the respective receptor.

etc.)

4.2

Molecular organization of colicin synthesis

Col plasmids code obligatorily beside colicins for one or, more often, two other types of proteins: an immunity protein (Sagik et al. 1983; Pugsley and Schwartz 1983), and a lysis protein (Pugsley 1984a). For example, the colicin operon of pColE1 carries three genes called the cea gene for the structure of colicin El, the i m m gene for the structure of the immunity protein, and the kil gene for the structure of the lysis protein (Fig. 1). The immunity protein cares for a specific protection of the colicinogenic cell against the self-produced colicin, but also against colicin of the same type acting from the environment. The lysis protein is a small lipoprotein, similar to the murein lipoprotein (Braun 1975), responsible for an easier release of the colicin synthesized from the producer cell. While the i m m gene is an element of all Col plasmids (some Col plasmids have even two i m m genes), the kil gene is present in small Col plasmids only (groups l a and/b). The kil genes of various Col plasmids show a high degree of homology. The organization of genes on the Col plasmids depends on the killing mechanism of their colicins, and in this way on the interaction between the colicin and its immunity protein. For colicins finding their lethal target in the plasma membrane (see parts 5.3.1 and 5.3.3), the i m m gene is transcribed constitutively, in the opposite direction than the structural gene for the colicin and the kil gene. For colicins having the nuclease activity, the i m m gene is a part of the transcription unit, is oriented in the direction of transcription of both other genes, and the intensity of its transcription is proportional to the transcription of these. Even in cells with the colicin operon switched off, some immunity protein synthesis continues, controlled by an independent weak promoter, which enables cells to survive in the medium with colicin spontaneously produced by a part of cells. The colicin synthesis is regulated by several mechanisms. The basic regulation, common to all colicins (except for colicins G, H and Js), is provided by the "SOS" system, which takes part in the control of gene expression, participating in the damaged DNA reparation. Under standard conditions, colicin synthesis is switched off in most cells of the population; it proceeds in a small part of population only, as a result of a random "spontaneous" activation of the "SOS" system.

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C O L I C I N S - EXOCELLUI.AR LETHAL PROTEINS O F E. c o l i - review

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Defects in the cell DNA result in the RecA proteinase activation (probably by an interaction with oligonucleotides, resulting from the DNA degradation), which breaks down and inactivates its own repressor, i.e. protein LexA, coded by a chromosomal gene. A positive feedback leads at first to a clear-cut expression of the gene for RecA protein, which is later suppressed by an increased synthesis of the LexA repressor, which represses its self-synthesis, ttowever, the LexA protein represses about 20 further chromosomal genes and also plasmid genes for the colicin synthesis. Preceding genes for the colicin synthesis, there are located two partly overlapping "SOS"-boxes, effectively suppressing the expression of genes for colicin, its immunity and lysis proteins (Lu and Chak 1996). This is the principle of the colicin synthesis induction by DNA attacking factors, like UV rays, mitomycin C, colicin E2, or some bacteriophages. However, an intensive synthesis of eoliein does not start -- in contrast to products of other "SOS"-regulated genes -- immediately, but after a so-called lag-phase. Perhaps this gives the cells an opportunity to repair their damaged structures, to repress the colicin operon again, and thus to divert the Kil protein lethal synthesis (Salles et aL 1987). The Kil protein of the pColA plasmid is synthesized as a preprotein, which is further modified (Cavard etal. 1985).

The colicin synthesis in a producer culture is increased as much as a thousand times by induction, after a lag-phase. Besides, in some colicins also more gentle regulation mechanisms participate in the management of this process. Further, colicin synthesis is regulated by a nonspecific catabolic repression. A low glucose concentration in the eolicinogenic cell medium leads to an increase of cAMP synthesis; cAMP is bound to the cAMP-receptor protein (CPR). This complex stimulates the expression of the structural gene for colicin by binding to DNA proximally from the promoter (Salles et aL 1987). The "stringent response" is the regulation of a stable mRNA synthesis according to the concentration of free amino acids in the cytoplasm. (Some genes are expressed, others are suppressed.) Perhaps, the response is mediated by the p p G p p (guanosine 5'-diphosphate 3'-diphosphate), whose increased concentration within a certain interval increases the E1 and E3 colicin synthesis 3-4 times at the transcription level (Lotz 1978).

Colicin production is also stimulated by anaerobiosis (~marda 1960). Under anaerobic conditions, transcription of the colicin gene is activated by the Fnr protein in some colicin plasmids. Conditions of anaerobiosis also shorten substantially the lag-phase, and the induced colicin synthesis is increased as much as 45 times in comparison with the aerobic conditions. The Fnr protein is synthesized constitutively, but is active under anaerobic conditions only. The binding of the active form of the Fnr protein proximally to the promoter initiates the transcription (Eraso and Weinstock 1992). An increased synthesis of colicin E1 during the late exponential and early stationary phases of the bacterial culture growth points to further possible regulation mechanisms of the colicin synthesis (Eraso et aL 1996). 4.3

Export of colicins from the producer bacteria

None of the colicins is transported specifically from producer cells. Colicins do not have any signal N-terminal sequences, and no export domains inside the colicin molecules have been found (Braun et al. 1994). Colicins are liberated semispecifically from producing cells, together with other proteins (Baty et al. 1987). Export of most colicins from the producer cells is greatly facilitated by the so-called lysis protein which causes also a release of the cell envelope (lysis) and death of the producer bacteria (Cavard et al. 1985). The expression of the kil gene for the lysis protein is regulated by the promoter, which is common with that for the colicin protein structural gene, and is therefore possible only following the induction of colicin synthesis in the cell. The lysis protein (after activation of the preprotein by acetylation) may be detected both in the plasma and the outer bacterial membrane (van der Wal et al. 1995b). The unusually stable signal sequence of the lysis protein is not degraded and contributes decisively to an efficient export of colicin from the producer cell (as of cloacin DF13 -- Luirink et al. 1992). Hence, the cell lysis and death results from the effect of both the lysis protein itself, and its signal sequence. It is probably a consequence of the intervention into the protein synthesis and into the transport of Mgz+ ions through the plasma membrane (Stegehius et al. 1995). The release of colicins synthesized is supported also by activation of the phospholipase A in the outer bacterial membrane, resulting in the production of lysophospholipids (Pugsley and Schwartz 1984). The absence of the kil gene in some Col plasmids (group I/) results in a markedly less efficient export of the given colicins. There is a lack of knowledge concerning the mechanism of release of colicins from producer ceils lacking the kil gene; it is perhaps a process of the secretion type.

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INTERACTION OF COLICINS WITH BACTERIAL CELLS

The inhibitory effect of colicins on bacteria is limited to sensitive strains of the family Enterobacteriaceae. This limitation results from the character of the interaction between the colicin and the bacterial cell, requiring the presence of a specific colicin receptor in the outer membrane, and of a functional translocation mechanism between the outer and the plasma membrane of the bacterium. The action of colicin on a sensitive cell proceeds generally through three phases (see also part 2): (1) Binding to the specific receptor in the outer bacterial membrane. (2) Translocation through the cell envelope. (3) The lethal effect (interaction with the intracellular target). This three-step action is reflected in the general organization of the colicin molecule, which is formed by three functional domains (see Fig. 2): (1) The receptor domain, which is the middle part of the colicin molecule, responsible for the binding of colicin to the receptor (Uratani and Cramer 1981; Mock and Pugsley 1982). (2) The translocation domain, the N-terminal part of the colicin molecule, responsible for its translocation through the cell envelope (Pugsley 1984a). (3) The lethal domain, the C-terminal part of the colicin molecule, responsible for the lethal effect, but also for the interaction of colicin with the immunity protein (Baty et al. 1988). colicin A colicin E1

colicin N

I 1. I

'

pyocin S2-colicinE3

I

Fig. 2. Schematicdiagramto compare the functional domainsof colicinsA, El, N and of the chimeric protein pyocin S2-colicin F_.3;T -- translocationdomain, R -- receptor-bindingdomain, L -- lethal domain. The most specificis the functionof the receptor-binding domain, less specificis the translocation domain.The lethal (bactericidal)domainis common to several colicintypes.

Each of the functional domains of the colicin molecule is relatively independent, and by means of artificial combinations of gene parts for the individual domains it is possible to construct not only new types of colicins but also hybrid exoproteins from colicins and other baeteriocins. Thus it is possible to "plant" the receptor and translocation domains of pyoein (bacteriocin of P s e u d o m a n a s aeruginosa) into colicin E3 and thus to obtain a hybrid protein killing not only the strains sensitive to colicin E3, but also strains sensitive to the original pyocin (Kageyama et al. 1996). Similarly, it is possible to construct hybrid molecules between colicins and bacteriophage proteins (Jakes et al. 1988). The degree of strain sensitivity to a colicin is, above all, a function of the average number of receptors per cell. Hence, coliein sensitivity is a quantitative character. In accordance with the course type of the colicin effect on sensitive bacteria and with the functional organization of colicin molecules, three types of insensitivity against them can be distinguished. (1) Resistance, due to the absence of a functional colicin receptor. In experimental practice, a culture which grows on the agar plate in the presence of colicin without any apparent limitation, is considered as resistant. However, such growth may be achieved also by a densely inoculated culture, where 90-95 % of the cells were killed by colicin. (2) Tolerance, due to the absence of a functional colicin translocation system. A tolerant cell has, in contrast to a resistant cell, an entirely functional receptor. Nevertheless, it usually binds less colicin than a sensitive cell does (Nomura 1964; Hill and Holland 1967). (3) Immunity. The cell has a functional colicin receptor and translocation mechanism. However, the development of a lethal effect is hampered by the interaction of colicin with the immunity protein, which is synthesized by cells of the producer strain together with colicin (Jakes and Zinder 1974). Thus immunity is equivalent to insensitivity of the producer cell. Besides, special types of a lowered colicin sensitivityhave been described: (i) to colicinM in cells with the tolM phenotype (Schalleret al. 1981); (ii) to cloacinDF13 (Wooldridgeand Williams1991); (iii) a nonspecificallylowersensitivityto

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co|icin B and probably to all membrane-depolarizing eolicins of the p a c B phenotype, associated with the resistance to tellurite in plasmids of the incompatibility group II (Whelan et aL 1995); (iv) to colicins E2, E3, D, la and lb in cells of the tolZ phenotype (Matsuzawa et al. 1984), and (v) to colicin E2 in cells w~th a dominant cet mutation, reducing the sensitivity to it by the accumulation of the Cet protein in ihe plasma membrane (Drury and Buxton 1988). The nature of these types of insensitivity or lowered sensitivity to colicins is not actually known.

5.1 Colicin-receptor binding Similarly as with bacteriophages, colicin binding to the sensitive cells is mediated by specific receptors in the outer bacterial membrane. These receptors serve primarily a very specific binding (with KD = 0.1 ~mol/L) of other, physiological, ligands, important for the bacteria/cell, such as of vitamin B12, or Fe 3§ in the complex form of siderophores. These are, however, secondarily "misused" by bacteriophages and colicins (Di Masi et al. 1973). A list of currently known E. coli receptors, used by colicins, and of their primary bond specificity, is presented in Table II. Table II. Survey of the known receptors and porins ofE. coli used by colicins, and their physiological specificity Protein of the outer membrane

Colicins using the protein as receptor

cooperating porin

BtuB

A, E l - E 9

FepA

B, D

Fiu Cir Tsx FhuA

G, II Ia, Ib, Q, S1, (also microcins V and B17) K, 5, 10 M

OmpF

N

OmpA IutA FyuAa TolC PhoE OmpC

U cloacin DF13 pesticin I El, 5, 10

A, E2-E9, I4, $4, U, cloacin DF13, bacteriocin 28b K, L, bacteriocin 2.8b

Bacteriophages using the protein as receptor

Physiological ligands and other functions

BF23

vitamin BI2 and other corrinoids 2,3-dihyd roxybe nzoyiserine enterochelin 2,3-dihydroxybenzoylserine 2,3-dihyd roxybe nzoylse rine

T6, Oxl, K9, II8 T1, T5, q~80, UC-1

nucleoside-specific porin ferrichrome, albomycin (analogue of ferr/chrome) nonspecific porin

Tula, TP1, TP2, SO108 Ox2, K3, MI

aerobactin yersiniabactin N N

TC45 T4, SS1, Tulb, TP2, Mel, Pa-2

anion-selective porin nonspecific ix)fin

a'llaough this receptor was described in Yersiniapestis, some strains o f E, coli are also sensitive to a pesticin (Ferber et al. 1981).

Binding of a colicin to its receptor is a typical protein-protein interaction, not requiring energy from the cell. It requires only appropriate ionic conditions, permitting an active conformation of both proteins. It proceeds with the same dynamics at 4 and 37 ~ The binding capacity of plasmolysed bacterial cells is, however, greatly decreased. In colicins of group A their interaction with specific receptors requires a cooperation of proteins of the outer membrane -- with porins OmpF, OmpA, OmpC, or with protein TolC. These must be, in some cases, also associated with lipopolysaccharides, as is the case, e.g., in colicins A and N or U (El Kouhen et al. I~Y)4;~majs et al. 1997). Cooperation with porins is not known for the B-group colicins. A certain analogy may be seen in the dependence of colicins 5 and 10 on ToIC (Braun 1995). Different colicins, which use the same receptor, usually distinguish different binding epitopes in it (Mock and Pugsley 1982; ~marda and ~ev~ov~i 1988). It can be assumed that sequences of these binding epitopes are much more conservative than the other sequences of receptors, exposed on the surface of the outer membrane. For instance, in the BtuB receptor, the binding domain of colicin E1 is quite distant from the cluster of epitopes for the other E colicins. In this cluster, the epitopes for colicins E2 and E3 are nearly identical. A bond of central sequence of the colicin molecule to the receptor probably introduces its conformation change, leading to the interaction of its N-terminal portion with the translocation system

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proteins. Colicins with the nuclease activity, appearing in the form of heterodimers with corresponding immunity proteins, split them upon binding to their receptors. The second group of outer membrane proteins, which interact with some colicins and participate in their translocation through it, are the porins. Porins are oligomeric transmembrane proteins forming "channels" through the outer membrane. E. coli porins are summarized in Table II. Some porins, such as Tsx or OmpF, are considered as direct receptors of colicins. Also other porins of the outer membrane are able to bind colicins to a certain limit (Kadner et al. 1979). Their role in colicin reception appears auxiliary. At least some of these porins can partly substitute for each other; e.g., colicin N binds not only to the OmpF porin but also to OmpC and PhoE (Evans et al. 1996). It seems that the group A colicins are able to use porins to overcome the barrier of the outer bacterial membrane (Lazdunski 1995). A point mutation, narrowing down the OmpF porin channel, causes the N-colicin resistance (Jeanteur et al. 1994). The group B colicins need probably receptor channels, which open by energization, for their penetration through the outer membrane (Braun 1995). 5.2

Colicin translocation through the cell envelope

There are two ways (two protein systems) for colicin translocation through the cell envelope to be used: Ton and Tol (Fig. 3). The Ton system is formed by proteins TonB-ExbB-ExbD (Kadner 1990; Braun et al. 1991). The Tol system includes proteins T o l A - T o l B - T o l O - T o l R (Lazzaroni et al. 1995). The Ton system ensures, e.g., transport of vitamin B12 and of colicins of group B. For the Tol system no "natural" substrates are known: it transports group A colicins, and F-pilus-dependent phages. Nevertheless, it may provide some primary transport function. TonB and ExbD, as well as TolA and TolR, are proteins anchored by their N-ends in the plasma membrane, TolO and ExbB are integral membrane proteins and TolB, which does not have any counterpart in the Ton system, is mainly localized in the periplasm. The TolA protein is analogous to the TonB protein, but they cannot substitute for each other. TolO is functionally interchangeable with ExbB, and TolR with ExbD. The TolC protein, needed for translocation of colicins El, 5 and 10, is not a part of the transcription group tolQRAB. Also its location in the outer membrane corresponds more to a receptor protein. Nevertheless, its receptor function has not been proved yet.

I..i .............. .

.

[

.

.

.

.

.

.

.

.

.

.

.

.

.

.

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.

.

.

....

,. .

]

121111111221122212111122112221 i122111 21122211221 [ pGI ps

[ExbD ~b~,~. '

Imm] [TolRJ~

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Fig. 3. Scheme of the E. coli bacterial envelope and its Ton and Toi components; OM -- outer membrane, PS -- periplasmic space, PG -- peptidoglycan layer, PM -- plasma membrane. See text for the explanation of protein abbreviations. The point is whether or not the peptidoglycan layer is uninterrupted by translocation complexes (at the contact sites of the outer and the cytoplasmic membrane).

The Tol complex is indispensable for the integrity of the bacterial cell envelope. Mutations in the tol genes result in the so-called tolerant phenotype (Clowes 1965; Nomura and Witten 1967), i.e. in a decreased cell sensitivity to colicins, but also in an increased permeability of the outer cell membrane to dyes, enhanced cell sensitivity to detergents, the loss of periplasmic proteins (in mutants tolA, tolB, andpal only), and defects in cell division and conjugation (Nagel de Zweig and Luria 1967; Threlfall and Holland 1970). Tolerance to colicins E brings about, at the same time, a decrease of the cell binding capacity for them. Mutation in the pal gene confers a similar phenotype, though the cell sensitivity to colicins has not been changed. The Pal lipoprotein is located in the outer membrane where it mediates a noncovalent connection of the TolB protein to its peptidoglycan (Bouveret et al. 1995). This indicates that Pal is an element of the Tol system. Using the TolB and Pal proteins, the Tol complex spans the entire system of the cell envelope. It is interesting that mutations in genes tol (including tolC) and pal cause the same tolerant phenotype while colicin transloeation depends on various combinations of functional Tol proteins.

The number of receptors per one sensitive bacterium ranges from 102 (e.g., BtuB) up to 105 (e.g., OmpF) copies. Nevertheless, the import of colicins is not limited simply by the number of recep-

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tor molecules on the cell surface but rather by the number of receptors associated with translocation complexes: e.g., the Tol system occurs in 400-1000 copies per cell only (Duch6 et al. 1995). Therefore, it appears that the attachment of many colicin molecules to specific receptors of the outer membrane does not necessarily result in their translocation through the cell envelope; a part of colicin molecules adsorbed are probably not transported to their lethal target (~marda 1975) although both transport systems interact with several different receptors. Some receptors cooperate alternatively with both translocation systems; in such cases, the transport of "natural" substrates is accomplished by the Ton system only, while colicins are able to make use of both Ton or Tol systems for their translocation. It seems that receptors for colicins, together with proteins of the translocation complexes, are not distributed at random over the cell envelope but are largely localized in the regions of contact of the inner and outer membrane. Their occurrence in these regions may be increased by their pre-incubation with colicin A (Guihard et al. 1994). Translocation of a colicin molecule is followed by rearrangement of its tertiary structure. Colicin A "unfolds" its structure during the translocation (releasing its tertiary structure), and its denaturation before translocation reduces the time necessary for the development of its lethal impact (B6n6detti et al. 1992). This, however, does not hold for all colicins, e.g. for colicin M. 5.3

Lethal effect of eolicins

The lethal effect of colicins is unusually pronounced. Studies of inhibition of sensitive bacteria by colicins indicate that a single one (Nomura 1963) or a few colicin molecules (Pattus et al. 1990; ~marda and Damborsk~ 1991) are sufficient to kill a sensitive bacterium. The bactericidal effect of colicins is preceded by a bacteriostatic one (~marda 1965; ~majs 1995). The duration of the bacteriostatic phase is an inverse function of the colicin multiplicity. Each cell binding colicin loses immediately its ability to divide but survives temporarily. In a population of sensitive bacteria, the colony-forming ability of cells decreases according to single-hit kinetics (Jacob et al. 1952). It is followed, with a few minutes' delay, by the lethal effect showing again the single-hit kinetics. (Within a 2-min exposure, colicins E1 and U show a transient fluctuation of the hit kinetics: a partial reversion of the adsorption and of the lethal effect -- Hej~itko and ~marda, in press). Through the effect of colicins on ceils in its bacteriostatic phase, aberrant forms of them are formed, such as fibres (e.g., by colicin E2 -- ~majs 1995). The killed cells lyse later on. The high efficiency of colicin killing is partly reduced by the fact that far not all colicin molecules bound to their receptor get associated with a translocation complex, and remain exposed to the influence of proteinases on the bacterial surface, which reverts the first, bacteriostatic, phase of their inhibitory effect (Nomura and Nakamura 1962; Cavard and Lazdunski 1990; see part 5.2). Poreforming colicins of both groups A and B keep their contact with the receptor during their channel formation through the plasma membrane. Therefore, their inhibitory effect may be eliminated by trypsin, which degrades parts of their molecules exposed on the cell surface, during a limited time interval. Similarly, sensitive bacteria in this inhibition phase may be revived by a colicin antibody (~marda 1965). Colicins kill sensitive bacteria in different ways. The most frequent way is the formation of ion channels in the plasma membrane, resulting in the depolarization of the membrane. Less frequent is the nuclease activity of colicins and least frequent is degradation or inhibition of synthesis of wall peptidoglycan. The nuclease activity can be directed against DNA (functionally eliminating the chromosome), or against 16S-rRNA (functionally discarding ribosomes). The given effect of the bactericidal domain of colicins is universal -- with the exception of colicin M and pesticin I. Colicins are able to insert into different membranes, to degrade any doublestranded DNA, or to inhibit protein synthesis on 70S or 80S ribosomes (Suzuki 1978). The activities of membrane-depolarizing and nuclease colicins have been verified in vitro experiments, too. We can only anticipate the mechanism of action of some further colicins (G, H). Some colicins had ceased to be available before the mechanism of their action could be explained (colicins C and P). The mechanism of action of some colicins (Js, Q) is still not understood. 5.3.1

Colicins depolarizing the plasma m e m b r a n e

Most of colicins are known to depolarize the plasma membrane (Jacob et al. 1952; Parker et al. 1989). The best understood members of this group are colicins A, El, B, Ia, Ib, K, N, U, 5, and 10, less known in this group are colicins S1 and $4 (Table I). The C-terminal domain of eolicins A and E1 is formed by ten 0t-helices, eight of which are amphiphilic, and two hydrophobic. The active domains, creating pores, represent two sequentially distinct types which can form a membrane channel permeable to ions, and thus depolarize the plasma membrane.

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The hydrophobic helices in aqueous solution are surrounded by amphiphilic chains. Molecules of colicins are therefore well soluble in water. During contact with the lipid bilayer of the membrane, the hydrophobic helices are inserted into it while the hydrophilic ones remain on the membrane surface (Duch6 et al. 1996). The insertion into the plasma membrane is oriented; it is possible only from its positively charged side; i.e. from the periplasmic space. Colicin channels alternate between the open and the closed form in a voltage-dependent manner. The transition of the channel to the open form is due to a dramatic rearrangement of the tertiary structure of the colicin channel domain. However, the exact mechanism is unknown. During the opening of the colicin Ia channel, a part of the eolicin molecule, at least 68 amino acid residues long, is translocated through the plasma membrane (Quiet al. 1996). The colicin A open channel tends to a decrease of the membrane potential from -165 mV (at the electronegative inner surface) to -85 mV (at pH 6.8), resulting in its closure (Bourdineaud et aL 1990).

The decrease of the membrane potential itself need not result in the cell killing effect of colicin, though it causes its energetic collapse. Thus it is necessary to look for another explanation of killing: depletion of K § inhibition of establishing a proton gradient, etc. Colicins G and H attack the plasma membrane, too, though probably in a different way. They cause a dramatic lysis of the wall-less L-forms of the sensitive cells (~marda and Taubeneck 1968). 5.3.2

Nuclease colicins

Colicins may affect sensitive bacteria as nonspecific DNA endonucleases (e.g., colicins E2, E7, E8, E9 -- Nomura 1963; ~marda et al. 1990), or as specific 16S-rRNA-endonucleases (coficins E3, E4, E6, cloacin DF13), breaking off a fragment of 49 nucleotides from the 3'-OH end of 16S-rRNA. This 16S-rRNA participates in the construction of the aminoacyl site on the 30S-ribosomal subunit of the 70S-ribosome (Konisky and Nomura 1%7; Lasater et al. 1989). Ribosomes, affected by colicins E3, E4, E6 or by cloacin DF13, are not further able to bind either mRNA due to the elimination of the sequence complementary to its Shine-Dalgarno sequence, or the aminoacyl-tRNA complex. In such a way, they block protein synthesis on ribosomes 70S (and 80S). It is interesting that the separation of colicin E3 from its immunity protein increases the efficiency of its h~ vitro effect, while decreasing its in vivo effect (Hirose et al. 1976). This can be interpreted as evidence of degradation of isolated eolicin molecules on the surface of sensitive bacteria. The immunity protein probably protects the colicin from degradation by proteinases. Colicins D and E5 also inhibit protein synthesis in sensitive cells but probably by a different mechanism. Their C-terminal parts are sequentially related neither to colicins E3, E4 or E6, nor to cloacin DF13. The DNAase colicins induce both the lytic cycle of bacteriophage in a lysogenic system, and the colicin synthesis in a colicinogenic one. 5.3.3

Colicin M and pesticin I

Colicin M inhibits synthesis of the wall murein and of lipopolysaccharide O by interference with the restitution of bactoprenyl phosphate (Schaller et al. 1982), and thus converts sensitive bacteria into spheroplasts, which lyse with a short delay. Pesticin I hydrolyzes murein and converts sensitive cells into spheroplasts (Ferber and Brubaker 1979; VoUmer et al. 1997).

6

I M M U N I T Y TO COLICINS

The effect of every colicin is blocked in producer ceils by a specific interaction with the relevant immunity protein (Konisky 1978). The specificity of this interaction is used for a more precise differentiation of colicins with the same receptor specificity (see part 4.1). The differences in the lethal effect mechanism of colicins are also related to differences in the colicin-immunity protein interaction. The immunity protein of pore-forming colicins is located in the plasma membrane of producer strain cells, and thus protects them from the exogenous colicin effect. Activity of the endogenous colicin is reliably suppressed by the "reversed" membrane potential (Lazdunski et al. 1988, see part 4.3.1). The immunity of producer strains of these colicins may be overcome by high doses of the exogenous colicin (like colicin N). It is also possible to construct strains devoid of their ability to synthesize the immunity protein. The colicin A immunity protein reacts with the hydrophobic helices of colicin A lethal domain

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(G61i and Lazdunski 1992; Espesset et al. 1996). It has four transmembrane helices, and this holds probably for all sequentially related immunity proteins of colicins B, N and U. The immunity protein of colicin E1 has a different structure with three transmembrane helices (Song and Cramer 1991), which is probably also the case of immunity proteins of colicins Ia, Ib, 5 and 10. Nuclease colicins (like E2 and E3) which operate within the cytoplasm, get associated with their immunity proteins immediately after having been synthesized which protects their producer strain cells from both exogenous and endogenous colicins. The immunity protein of colicin E7 has four antiparallel ix-helices with a negatively charged variable region, which interacts with the lethal domain of the colicin (Chak et al. 1996). The immunity protein is split off from the colicin as late as during the binding to the receptor of a sensitive cell. In the producers of these colicins it is not possible to overcome the immunity by high doses of colicins or to prepare strains which would not be able to synthesize the immunity protein (Pugsley 1984b). The immunity protein of colicin M is anchored by its N-end in the plasma membrane of producer cells. It protects them against the effect of an exogenous colicin M by its periplasmic part which forms a complex with it (01schl~iger et al. 1991). Also the pesticin I immunity protein is anchored in a similar way. Again, it is its periplasmic part that is decisive for the immunity of the producer cell (Gross and Braun 1996; Pilsl et al. 1996).

INHIBITORY EFFECTS OF COLICINS ON BACTERIA D E P R I V E D OF T H E CELL WALL, AND ON E U K A R Y O T I C CELLS Colicins are able to kill, besides the standard sensitive bacteria, also their stable L-forms of the protoplast type, i.e. bacterial cells deprived of their outer membrane and of the peptidoglycan layer, and covered with a single plasma membrane (~marda and Taubeneck 1968). By this transformation, the sensitivity to some colicins (G and H) is greatly increased, not changed to others, while being decreased to some others (e.g. to colicins E2 and E3). These phenomena indicate the varying degrees of importance of the receptors for the mechanism of various colicin effects. The lethal effect of colicins on the L-forms of bacteria is direct (one-phase); it cannot be inhibited by trypsin (~marda and Taubeneck 1968). The number of colicin molecules that can be bound by the L-form of a sensitive bacterium, is higher than in the standard cell equipped with a wall (~marda and Schuhmann 1979). The binding of colicin onto the protoplast-type cell requires energy of its membrane. This finding, together with the results showing that also in a population of rods of a resistant strain, 90 % of the bacteria are inhibited by colicin, in spite of the fact that they dispose of no functional receptor (~marda 1992a), initiated the research of the inhibitory action of colicins also on eukaryotic cells, i.e. on cells of species quite remote from the family Enterobacteriaceae. E.g. colicin E3 inhibits: (i) mouse fibroblasts L (LDs0 = 105 lethal units of colicin per cell -~marda et al. 1978); (ii) human tumor cells HeLa (LDs0 = 3"103 LU per cell -- ~marda and Obdr~-~lek 1977), (iii) mitogenic activation by concavalinA in T-lymphocytes (Viklick~ et al. 1979); it also increases the redox activity of peritoneal leukocytcs (Lokaj et al. 1982). The degree of sensitivity of different cells to colicins is variable. Nevertheless, sensitivity of tumor cells to colicins is generally higher than that of standard cells while different tumor lines differ again in their sensitivity to different colicins (~marda 1992b). E.g. colicin A kills 53-58 % lymphoma cells, as well as lymphosarcoma, and plasmocytoma cells in cultures (~marda and Oravec 1993). The antitumor effect of colicins was confirmed in vivo: colicins prolonged the survival period of mice with plasmocyloma from 44 to 63 d, and of mice with HK-adenocarcinoma from 23 to 28 d (Fig. 4). Tumor necrosis has been also proved by histological tests (Fig. 5 -- ~marda 1983). These results verify the universal effect of the lethal domains of both membrane-depolarizing and nuclease colicins. Lethal targets for these colicins, i.e. the plasma membrane, DNA, and 16S-rRNA, are present also in the eukaryotic cells, and receptors of the bacterial outer membrane are not necessary to mediate their lethal effect.

8

E V O L U T I O N OF COLICINS A N D OF Col PLASMIDS

All colicins have an analogous domain structure. Differences between them are given by the different properties of their individual domains: the receptor, the translocation, and the lethal ones. Comparing the amino acid sequence of the individual domains between colicins, we can often identify

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identical sequence motifs. For instance, colicins E2 and E3, B and D, K and 5 have identical (or very similar) receptor domains; translocation domains of the A group colicins are functionally similar or

f

Fig. 4. Pathological section of the mouse llK-adenocarcmoma; top: control tumor treated with 8.75 lug dextran C daily for 24 d;

bottom: tumor treated with 0.875 lug colicin E3 daily for 24 d.

identical, the lethal domains are very often alike. From the Table I it is evident that 29 different types of bacteriocins are constructed from 13 types of different receptor domains with different specificity, 2 types of translocation domains, and 7 - 8 types of lethal domains. Theoretically, by a simple combination of the already known domains, 182-208 different types of colicins (each containing three domzin.~) could be constructed. The situation is further complicated by the dependence of some colicin effects on porins of the outer membrane, or on proteins TolB and TolC.

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The producer cells may carry genes for two colicins on one Col plasmid (as colicins B and M; Fredericq and ~marda 1970), or additional genes for immunity proteins; thus pColE3-CA38 and pColE6-CT18 have an additional gene for the immunity protein of the E8 colicin. Producer cells can, at the same time, carry several different Col plasmids, the selection of which is directed by their relevance to incompatibility groups (Fredericq 1948; Horfik 1994). All these findings suggest the possibility of intra- and intergenic recombinations during the evolution of colicin genes and of entire Col plasmids. The evolutionary differentiation of genes for colicins, for their immunity and lysis proteins, as well as the evolution of Col plasmids themselves, were and are due to mutations, transpositions and recombination processes (Lau and Condie 1989; James et al. 1996).

Fig. 5. Itistologicalsection along the largest circumferenceof extirpated mouse llK-adenocarciaomatreated for 24 d; top: with 8.95 lag human serum albuminedaily;,bottom: with 0.875lagcolicinE3 daily.Livingtissueviolet, necrotic massesred.

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However, the question of the origin or the evolutionary ancestors of colicins and Col plasmids still remains open. The sequence analysis of biomembrane-depolarizing and nuclease lethal domains of colicins did not show any relation to the cell proteins known. It appears, therefore, that colicins originated independently of them (Braun et al. 1994).

9

P R A C T I C A L APPLICATIONS OF COLICINOGENY A N D COLICINS

The start of colicin research was not motivated by any concrete needs. The colicins were and are studied simply because they exist. Nevertheless, with the increasing accumulation of information, ideas of possible applications of them arose, too. Colicins are invaluable tools for studies of cell functions. Research into the export of colicins from the producer bacterial cells and import of them into the sensitive ones enables to follow the molecular mechanisms of these processes. The new, highly efficient, cephalosporin antibiotics (substituted, e.g., by pyridone) are transported into bacteria by a similar mechanism as some colicins, with participation of the Ton system (Tatsumi et al. 1995). In bacteria, equipped with a recombinant DNA which ensures the production of eukaryotic proteins, their export from the producer cells, induced by the expression of the colicin lysis protein, is being studied in gene engineering projects (van der Wal et al. 1995a). Research is also aimed at the Tol system and its role in the determination of the bacterial envelope, as well as at the translocation of colicin molecules across the two biomembranes. Colicins serve also as a model of insertion and formation of ion channels in the plasma membrane. The highly specific inhibition of protein synthesis by RNAase colicins helps to clear up the general translation mechanism. The interaction of colicins with immunity proteins, as well as with receptors, permits to study protein-protein interactions. Research into Col plasmids finds its applications also in gene engineering. Many vectors have their origins of replication derived from Col plasmids, and/or from related plasmids (pBR322, pBCSK). The recombinant plasmids with the desired genes can be stabilized in host bacteria using the colicin and its immunity protein genes system. Plasmids can also be constructed with the sole colicin structural gene, with the gene for the immunity protein inserted into the chromosome which can then replicate in the mother strain only. Such an approach can block the horizontal transfer of recombined plasmids (Mathildah et al. 1996). It seems possible to allot a new target specificity to the lethal domains of colicins. Colicin production is used for epidemiological typing of bacterial strains, such as Shigella sonnei (Abbot and Shannon 1958; Hor~ik 1994). Since the sensitivity to colicin Js is always associated with the entcroinvasive serotypes E. coli (Abbott and Shannon 1958; Hor~k and Sobotkov~ 1988), it can be used as a preliminary test for virulence of this species isolates. The importance of the presence of colicinogenic E. coli bacteria in the human intestinal tract has been proved by successful therapeutic and preventive exchanges of the original bacterial strains E. coli in the intestine by the preparation MUTAFLOR | This strain produces a not yet exactly defined bacteriocin (or a combination of bacteriocins), marked as colicin X (Papavassiliou 1961). (This colicin is probably not identical with the current type colicin X which is identical with microcin B17.) Promising experiments, aiming at the use of the bactericidal effect of colicins for the treatment of coli infections in ophthalmology and dermatology had, unfortunately, to be interrupted before results suitable for publication have been achieved (J. Mach, personal communication).

10

CONCLUSIONS

The research into colicins has discovered laws of their synthesis, basic mechanisms of their molecular interaction with the bacterial cell, and the basic evolutionary principle of their molecular construction. The questions of the biological sense of colicinogeny and of its ecological importance still remain open, both at the bacterial population level, and at the level of interaction between colicinogenic bacteria and the host organism. Is it the matter just of a simple selective advantage of the producer cells over the sensitive ones in special situations of the life of coliform bacteria, or of a plasmidstabilizing system with discrete advantages in the bacterial population, or of a system, which uses the suicidal induction of the colicin operon expression to eliminate less apt bacteria? Research into colicins is the more attractive, the more it contributes, besides the understanding of the sense and essence of

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the phenomenon ofcolicinogeny itselL no doubt also to a better understanding of some general laws of biology. This review was supported by grant no. 310/98/0083 of the Grant Agency o f the Czech Republic.

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BATY D., LLOUBESR., GI2LI V., LAZDUNSKIC., IIOWARD S.P.: Extracellular release of colicin A is non-specific. EMBO Z 6, 2463-2468 (1987). Bf~NP.OEITIII., I.LOUB'ESR., I.AZDUNSrdC., L~'rrELn.:a L.: Colicin A unfolds during its translocation in Escherichia colt cells and spans the whole cell envelope when its pore has formed. EMBOJ. 11, 441-447 (1992). BEN-GuRION R., IIERTMANI.: Bacteriocin-like material produced by Pasteurella pbstis. J.Gen.Microbiol. 19, 289-294 (1958). BOURDINEAUD J.P., BOULANGERP., LAZDUNSKI C., LEa~LLIER L.: In vivo properties of colicin A: channel activity is voltage dependent but translocation may be voltage independent. Proc.Natdlcad.Sci.USA 87, 1037-1041 (1990). BOUVI.:R~'TE., DEROUICttER., RIGALA., LLOUBF.SR., LAZDUNSKIC., BI~Nt~DETFIII.: Peptidoglycan-associated lipoprotein-TolB interaction. J.BioLChem. 270, 11071-11077 (1995). BRADLEY, D.E.: Colicins G and II and their host strains. CanJ.MicrobioL 37, 751-757 (1991). BRADLEY D.E., HOWARD S.P.: A new colicin that adsorbs to the outer membrane protein Tsx but is dependent on the tonB instead of the tolQ membrane transport system. J.Gen.Microbiol. 138, 2721-2724 (1992). BRANDISII., ~MARDAJ." Bacteriocine und bacteriocin/ihnliche Substanzen. Gustav Fischer Verlag, Jena 1971. BRAUNV.: Covalent lipoprotein from the outer membrane of Eschen'ctu'a colt. Bioclu'm.Biophys,,lcta 415, 335-377 (1975). BRAUN V.: Energy-coupled transport and signal transduction through the gram-negative outer membrane via the T o n B ExbB-Exbl) dependent receptor proteins. FEMS Microbiol.Rev. 16, 295-307 (1995). BRAUNV., G(~NTERK., I-IANTKEK.: Transport of iron across the outer membrane. BioLMet. 4, 14-22 (1991). BRAtm V., PILSL 1t., GROSS P.: Colicins: structures, modes of action, transfer through membranes, and evolution. Arch.Microbiol. 161, 199-206 (1994). CAVARDD., LAZDUNSrOC.: Colicin cleavage by OmpT protease during both entry into and release from Escherichia colt cells. 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