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Biosynthesis of bacteriocins in lactic acid bacteria

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Biosynthesis of bacteriocins in lactic acid bacteria
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  Antonie van Leeuwenhoek 70:113-128, 1996. ~) 1996 Kluwer Academic Publishers. Printed in the Netherlands. 113 Biosynthesis of bacteriocins in lactic acid bacteria Ingolf E Nes*, Dzung Bao Diep, Leiv Sigve H~varstein, May Bente Brurberg, Vincent Eijsink Helge Holo Laboratory of Microbial Gene Technology, Department of Biotechnological Sciences, Agricultural University of Norway, P.O. Box 5051, N-1432 ~s, Norway *author or correspondence) Summary A large number of new bacteriocins in lactic acid bacteria LAB) has been characterized in recent years. Most of the new bacteriocins belong to the class II bacteriocins which are small 30-100 amino acids) heat- stable and commonly not post-translationally modified. While most bacteriocin producers synthesize only one bacteriocin, it has been shown that several LAB produce multiple bacteriocins 2-3 bacteriocins). Based on common features, some of the class II bacteriocins can be divided into separate groups such as the pediocin-like and strong anti-listeria bacteriocins, the two-peptide bacteriocins, and bacteriocins with a sec- dependent signal sequence. With the exception of the very few bacteriocins containing a sec-dependent signal sequence, class II bacteriocins are synthesized in a preform containing an N-terminal double-glycine leader. The double-glycine leader-containing bacteriocins are processed concomitant with externalization by a dedicated ABC- transporter which has been shown to possess an N-terminal proteolytic domain. The production of some class II bacteriocins plantaricins of Lactobacillus plantarum Cl 1 and sakacin P of Lactobacillus sake) have been shown to be transcriptionally regulated through a signal transduction system which consists of three components: an induction factor IF), histidine protein kinase HK) and a response regulator RR). An identical regulatory system is probably regulating the transcription of the sakacin A and carnobacteriocin B2 operons. The regulation of bacteriocin production is unique, since the IF is a bacteriocin-like peptide with a double-glycine leader processed and externalized most probably by the dedicated ABC-transporter associated with the bacteriocin. However, IF is not constituting the bacteriocin activity of the bacterium, IF is only activating the transcripion of the regulated class II bacteriocin gene s). The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins. Introduction The antimicrobial effect of lactic acid bacteria LAB) has been appreciated by man for more than 10 years and has enabled him to extend the shelf life of many foods through fermentation processes. The major preservative effect of LAB is due to their production of lactic acid which results in a concomitant lowering of pH. For a long time it has been known that many LAB also produce additional antimicrobial compounds and among these the antimicrobial ribosomally synthesized peptides, generally termed bacteriocins, have received special attention, from both scientific and the food- industrial communities. Since the late 1920s and early 1930s, when the discovery of nisin initiated the investigation of pro- teinaceous antimicrobial compounds from LAB, a large number of chemically diverse bacteriocins have been identified and characterized, particularly in recent years. Nonetheless, we can observe common traits which justify their classification into just a few class- es Klaenhammer, 1993). On a sound scientific basis, three defined classes of bacteriocins in LAB have been established, class I: the lantibiotics; class II: the small heat-stable non-lantibiotics and class III: large heat- labile bacteriocins Table 1). A fourth class of bacte- riocins has also been defined, which contains bacteri- ocins composed of an undefined mixture of proteins, [17]  114 Table 1. Classification of LAB Bacteriocins Class I. Class II Class IlL Lantibiotics Small heat stable non lantibiotcs IIa Pediocin-like bacteriocins with slxong antilisterial effect IIb Two-peptide bacteriocins IIc sec dependent secreted bacteriocins Large heat labile proteins lipids, and carbohydrates. The existence of the fourth class was supported mainly by the observation that some bacteriocin activities obtained in cell-free super- natant, exemplified by the activity of Lactobacillus plantarum LPCO10, were abolished not only by pro- tease treatments, but also by glycolytic and lipolytic enzymes (Jim6nez-Diaz et ai., 1993). However, such bacteriocins have not yet been characterized adequate- ly at the biochemical level and the recognition of this separate class therefore seems premature. Indeed, the experimental data suggest that these complex bacterio- cinogenic activities may be artifacts caused by interac- tion between constituents from the cells or the growth medium and the undefined bacteriocin activities are likely to be regular peptide bacteriocins. This view is strongly supported by experiments showing that proper purification of such activities indeed leads to the iso- lation of regular peptide bacteriocins (Jim6nez-Diaz et al., 1995). Bacteriocin activity is frequently found associated with large aggregates in cell free extracts. These aggregates include not only proteinaceous mate- rial but, most probably, also lipids and other macro- molecules which could affect the bacteriocin activity. Several bacteriocin containing aggregates are resolved into simple peptide bacteriocins by purification. Var- ious enzymatic treatments may affect the bacteriocin activity of these crude complexes. LAB bacteriocins of class I and II are by far the most studied because they are both the most abundant ones and the most prominent candidates for industrial application. Members of the two classes are general- ly clearly different, both in terms of the structure of the bacteriocin itself and in terms of the machinery involved in production and processing. A few interme- diate cases have been observed, namely a few lantibi- otics (class I) that are secreted by mechanisms charac- teristic for class II bacteriocins (Piard et al., 1993; Ross et al., 1993; H,~tvarstein et al., 1994, 1995). The present review focuses on class II bacteriocins only. Class I bacteriocins have recently been reviewed extensively by others (De Vos et al., 1995; Jack et al., 1995; Sahl et al., 1995; Konings & Hilbers, 1996). Diversity of class II bacteriocins A large number of class II bacteriocins has now been biochemically characterized mainly due to the develop- ment of efficient and standardized protocols for purifi- cation of these hydrophobic and cationic peptides. The availability of biochemical characteristics of a large number of class II bacteriocins now permits a sub- grouping of many of these compounds. However, one should keep in mind that the subgrouping of bacteri- ocins is just a way to organize our present knowledge in a functional way, and future research will certainly change our present concepts of the class II bacteriocins. One major subgroup of bacteriocins shows very strong antilisterial activity. Members of this subgroup (class IIa) are found in a wide variety of LAB includ- ing Pediocicoccus (Henderson et al., 1992; Marug et al., 1992; Motlag et al., 1992; Nieto Lozano et al., 1992; Ray, 1992), Leuconostoc (Hastings et al., 1991; Hechard et al., 1992), Lactobacillus (Holck et al., 1992; Larsen et al., 1993; Tichaczeck et al., 1992; Kanatani et al., 1995) and Enterococcus (Aymerich et al., 1996). The antilisterial bacteriocins share strong amino acid sequence homology (between 38-55 identity) which is most pronounced in the N-terminal part of the peptides (Aymerich et al. 1996). This sub- class of bacteriocins has been termed the pediocin- family after the first and most extensively studied example of this class, pediocin PA-1. A second subgroup (class IIb) contains bacteri- ocins whose activity depends on the complementary action of two peptides and several examples of such bacteriocins have been studied (van Belkum et al., 1991, Nissen-Meyer et al., 1992, 1993b; Allison et al., 1994; JimEnez-Diaz et al., 1995; Diep et al., 1996). It should also be mentioned that one example of a two- peptide lantibiotic has been characterized (Gillmore et al., 1994). All bacteriocins are synthesized with an N-terminal leader sequence and, until recently, only the double- glycine type of leader was found in class II bacte- riocins (see below) (Holo et al., 1991, Muriana & Klaenhammer, 1991; Klaenhammer, 1993; H,~tvarstein et al., 1994). However, it has now been disclosed that some small, heat stable, and non-modified bacteriocins are translated with sec dependent leaders (Leer et al., 1995, Worobo et al., 1995). Due to their similarity to [18]  the class II bacteriocins they should be included as a separate subgroup, the class IIc (Table 1). It has been suggested that a subgroup of thiol- activated bacteriocins (lactococcin B) should be included (Venema et al. 1994), but recent findings suggest that this subgroup should be excluded since oxidation of the sulphydryl group with other chem- icals did not interfere with its activity (Venema et al. 1995). It was also shown that when this cysteine residue was replaced by all other amino acids, only the posi- tive charged amino acids were reducing/abolishing the bacteriocin activity which suggests that the cysteine is note essential for the biological activity (Venema et al. 1995). The synthesis of the cationic peptide-bacteriocins rest upon a general genetic structure encompassing four different genes which encode the basic functions required for production of the extracellular antimicro- bial activity (Nes et al. 1995). These four genes are: 1) the structural gene encoding the prebacteriocin, 2) a dedicated immunity gene always located next to the bacteriocin gene and on the same transcription unit, 3) a gene encoding a dedicated ABC-transporter which externalizes the bacteriocin concomitant with process- ing of the leader, and 4) a gene encoding an accessory protein essential for the externalization of the bacteri- ocin, the specific role of which is not known. The four basic genes are organized either in one or two operons. In the lactococcin A system two operons are found (Holo et al, 1991, van Belkum et al. 1991, Stoddard et al., 1992) while the pediocin PA-1 system possesses one operon comprising the four genes involved in pro- duction of the active bacteriocin molecule (Marugg et al. 1991). In addition to the four basic genes, regulato- ry genes have been found associated with the genetic determinants of some class II bacteriocins (Diep et al., 1994, 1996; Axelsson Holck, 1995; Quadri et al., 1995a; Huehne et al., 1996; Brurberg et al., 1996). These findings are discussed separately below. The works of van Belkum et al., (1991, 1992) revealed that one Lactococcus lactic strain can pro- duce more than one bacteriocin. This particular Lac tococcus strain produces three plasmid-encoded bac- teriocins, two one-peptide bacteriocins and one two- peptide bacteriocin. Recent studies have shown that production of multiple bacteriocins by one organism is quite common. In Carnobacteriumpiscicola LV17B it has been shown that at least two different bacteriocins are produced, one is plasmid-encoded while the oth- er is found on the chromosome (Quadri et al., 1994; 1995a,b). The bacteriocin secretion system which was 115 physically located on the plasmid next to the bacteri- ocin gene, was also used by the chromosomally encod- ed bacteriocin. Lactobacillus plantarum C 11 also pro- duces multiple bacteriocins including two two-peptide bacteriocins and one one-peptide bacteriocin (Diep et al., 1996). In Lactobacillusplantarum C11 the bacte- riocin genes and their accessory genes are clustered on the bacterial chromosome. he bacteriocin and its gene The structural bacteriocin gene encodes a preform of the bacteriocin containing an N-terminal leader sequence (termed double-glycine leader) whose func- tion seems a) to prevent the bacteriocin from being biologically active while detained inside the producer, and b) to provide the recognition signal for the trans- porter system (see below). The double-glycine leader varies in length from 14 residues up to approximate- ly 30 residues (Klaenhammer, 1993, Hiivarstein et al., 1994). The consensus elements found in the double- glycine leader include the two glycine residues at the C-terminus of the cleavage site, conserved hydropho- bic and hydrophilic residues separated by defined dis- tance between the conserved residues. In addition the minimum length of the double-glycine leader seems to be 14 amino acids (Table 2A). Of the consensus residues in the leader, only the glycine at position - 2 residue is fully conserved. The mature bacteriocins identified so far vary in size from less than 30 residues to more than 100 residues in some cases. It should also be mentioned that colicin V (88 residues) ofE. coli can be formally classified as a class II bacteriocin (Fath et al., 1995, Hiivarstein et al., 1995). Recently, a second E. coli bacteriocin, microcin 24, can also be classified as a class II bacteriocin according to the nomenclature (O'Brian et al., 1996). The class II bacteriocins share a number of common features. They have a high content of small amino acids like glycine. They are strongly cationic, with pI's usu- ally varying from 8 to 11, and they possess a hydropho- bic domain and/or amphiphilic region, which may relate to their activity on membranes (Abee, 1995). As referred to above, a number of bacteriocins consists of two peptides. Both peptides possess a double-glycine leader and are encoded by individual, contiguous genes in the same operon. Both peptides are structurally indistinguishable from the one-peptide bacteriocins, however, both peptides are apparently required for activity, or for obtaining optimal activity. [19]  116 Table 2. Leader sequences found n Class II bacteriocins A ABC-transporter-dependent leaders (consensus sequence) Double glycine eaders: - - LS - - EL - - I - GG (14-30 residues) Consensus: - -*~ - ~ ~ * - -* - GG 9 Hydrophobic residues, DHydrophilic residues sec-dependent signal sequences Divergicin A: MKKQILKGLVIVVCLSGATFFSTPQASA Acidicin B- MVTKYGRNLGLSKKVELFAIWAVLVVALLLATA The antimicrobial activity of lactococcin G and prob- ably lactococcin MN is completely dependent on both peptides (Nissen-Meyer et al., 1992, van Belkum et al., 1991). On the other hand, one of the peptides of 9 he two-peptide bacteriocins plantaricin S and lactacin F possesses apparently some antimicrobial activity. However, the second peptide has a dramatic effect by enhancing and/or modifying the activity (Allison et ai., 1994, Jim6nez-Diaz et al., 1995). In the plantaricin S system the (a-peptide does not show any detectable bacteriocin activity while the bacteriocin activity of ~-peptide is enhanced approximately 50-fold by the a-peptide. Lactacin F activity is defined by the two functional tafA and lafX genes located next to each other. LafA is a bacteriocinogenic peptide by itself but upon addition of LafX the activity of LafA increases and the inhibitory spectrum expands. While most bacteriocins appear to be secreted by the sec- independent universal ABC-transporter sys- tem (see below), it has recently been shown that some bacteriocins do not possess a double-glycine leader sequence but are, instead, synthesized with a typi- cal N-terminal leader sequence of the sec-type. This is a new and very interesting feature, which demon- strates that bacteriocins can be secreted/processed by two different pathways. So far only two LAB bacte- riocins containing the sec-dependent signal sequences are known (Table 2B) (Leer et al., 1995; Worobo et al., 1995). he immunity protein and its gene Bacteriocin producers have developed a protection system against their own bacteriocin. This system is referred to as immunity. Each bacteriocin has its own dedicated protein conferring immunity, which is expressed concomitantly with the bacteriocin. In all bacteriocin operons studied so far, potential immu- nity genes have been identified next to and down- stream of the bacteriocin structural genes. While syn- thesis of extracellular bacteriocin requires a dedicat- ed secretion/processing system, the immunity protein is functionally expressed in the absence of transport and processing. This has been proven convincingly for a number of immunity factors including LciA and PedC of lactococcin A and pediocin PA-1, respec- tively (Holo et al. 1991, Venema et al. 1994, 1995a). When lactococcin A and its immunity gene (IciA) were cloned into lactococcin A sensitive strains, only LciA was functionally expressed while LcnA activity was not detected because the transporter/processing system was missing in the new host. The immunity proteins are fairly small, ranging from 51 to approximately 150 amino acids and the homology between various immunity proteins is sur- prisingly low when considering the similarity found between several bacteriocins (Aymerich et al. 1996, Holo 1996). This lack of similarity is particularly strong between the immunity proteins of the two iden- tical bacteriocins, sakacin A and curvacin A (Axelsson et al. 1993, Tichaczek et al. 1993), where the putative immunity proteins are 90 amino acids and 51, respec- tively. Most of the immunity proteins of the bacteri- ocins belonging to the pediocin-family do not share significant homology while these bacteriocins share 38-55 % identity (Aymerich et al. 1996). This obser- vation may suggest that no direct interaction occurs between bacteriocins and their immunity proteins. The putative immunity proteins of curvacin A and acidocin A (51 amino 55 amino acids respectively) are smaller than other immunity factors and computer- assisted amino acid sequencing analyses indicates that the N-terminal regions of both immunity proteins can form membrane spanning helices. Hydropatic profile analyses of some immunity proteins have revealed up to four putative transmembrane segments, which sug- gests that these immunity factors may integrate in the membrane of the bacteriocin producer (Fremaux et al. 1993). The immunity protein of lactococcin A (LciA) has been purified to homogeneity and in vitro experiments suggest that LciA does not interact directly with lac- tococcin A, although such experiments, of course, do not exclude that direct interaction can take place in vivo (Nissen-Meyer et al. 1993). It has also been shown that the immunity protein of lactococcin A is intracellular- [20]  117 DomalnB Proteolytic Membrane spanning ATP-binding I~-Domain ~ Domain -rJ~ Domain [ It ll || || | C I -i --150aa --800aa --250aa C = Cystaine containings motif ~ = ATP-binding motifs 0 H = Histidine containing motif ~ = Membrane helices Membrane Localization Exterior Interior Figure 1 The ABC-transporter of class II bacteriocins with double- glycine leaders. A: The organization of the domains of the transporter. B: The presumed localization of the domains in relation to the cyto- plasmatic membrane. ly located and between 50 and 90 of the immunity protein is found free in the cytoplasm (Nissen- Meyer et al. 1993, Venema et al. 1994). The work of Ven- ema et al. (1994) also suggests that the amphiphilic a-helix domain (residue 29 to 47) of the immunity protein of lactococcin A is embedded in the membrane with the C-terminal end directed towards the exteri- or of the cell. Free intracellular LciA is considered a reservoir to be used for the defense when needed. It has also been proposed that LciA is closely associated with a postulated bacteriocin receptor (Venema et al. 1994), however, the presence of such a receptor is still speculative and the molecular entity has yet to be iden- tified. The most intriguing and challenging problems in the field of bacteriocin research today have to do with the molecular mechanism behind the immunity of bacteriocins and questions related to the existence and identity of bacteriocin receptors. The ABC transporter As mentioned above, most bacteriocins are synthe- sized in a preform containing an N-terminal exten- sion, the so called double- glycine leader (Table 2A), first identified in lactacin F and lactococcin A (Holo et al., 1991, Muriana & Klaenhammer, 1991). The strong conservation of the cleavage site of the leaders strongly suggests a common processing mechanism for these peptides. The first evidence suggesting that ABC-transporters are required for extracellular acti- vation of class II bacteriocins was presented by Stod- dard et al. (1992), during studies of the lactococcin A, B and MN systems. In this study the genes of a dedicated ABC-transporter (LcnC) and its accessory protein (LcnD) were identified to be needed for pro- duction of active extracellular lactococcin A. Today it is well established that secretion of the double-glycine leader containing bacteriocins is mediated by a ded- icated transmembrane translocator belonging to the ATP-binding cassette (ABC) transporter superfamily (Gilson et al., 1987, 1990; Gillmore et al., 1990, 1994, Stoddard et al., 1992; Marugg et al., 1992; H,~tvarstein et al., 1995). The bacteriocin ABC- trans- porter gene is usually either part of the bacteriocin operon or found on a separate operon in the vicini- ty of the bacteriocin-containing operon. It has been generally recognized that all ABC-transporters con- tain two domains, a hydrophobic integral membrane domain and a cytoplasmatic ATP-binding domain (Fig- ure 1). By comparing the amino acid sequence of seven ABC-transporters dedicated to the transiocation of bacteriocins containing the double-glycine leader type with other ABC-transporters, a unique feature in the N-terminal part of the bacteriocin transporters has become apparent (Figure 1). It was noticed that the bacteriocin transporters carried an N-terminal exten- sion of approximately 150 amino acids which was also found in a few other systems including the c~- haemolysin transporter of Escherichia coli that of the competence system of Streptococcus pneumonia and in the transporters of some lantibiotics (Hhvarstein et al., 1995). However, the ABC-transporter of lantibiotics such as nisin, with a leader sequence different from the double-glycine type, does not have this extension. For some time the unanswered questions were: where, when and by which protease is the double-glycine lead- er removed? The observation suggested that the N- terminal extension of the transporter could be involved in the processing of the bacteriocins. Functional stud- ies of the N-terminal region of the ABC-transporter of lactococccin G were performed. The N-terminal extension of the transporter was cloned and expressed, and enzymatic studies were performed on the cloned polypeptide fragment in order to determine its enzy- matic role in the transport process. It was convincingly demonstrated that the N-terminal polypeptide was able [211
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