Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication

Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being
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   Antonie van Leeuwenhoek   81:  639–654, 2002.© 2002  Kluwer Academic Publishers. Printed in the Netherlands.  639 Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication Vincent G.H. Eijsink  1 , ∗ , Lars Axelsson 2 , Dzung B. Diep 1 , Leiv S. Håvarstein 1 , Helge Holo 1 &Ingolf F. Nes 1 1  Agricultural University of Norway, Department of Chemistry and Biotechnology, P.O. Box 5040, N-1432 ˚  As, Norway;  2  MATFORSK, The Norwegian Food Research Institute, Osloveien 1, N-1432 ˚  As, Norway ( ∗  Author for correspondence)Key words:  anti-microbial peptide, bacteriocin, immunity, lactic acid bacteria, pheromone, quorum sensing Abstract Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptidescalled bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (classII), the latter being the main topic of this review. During the past decade many of these bacteriocins have beenisolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have beenunravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closelyrelated bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for thesenarrow inhibitory spectra by producing several bacteriocins belonging to different classes and having differentinhibitory spectra. The latter may also help in counteracting the possible developmentof resistance mechanisms intarget organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using asecreted peptide-pheromonefor quorum-sensing. The sensing of its own growth, which is likely to be comparableto that of related species, enables the producing organism to switch on bacteriocin production at times whencompetition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocinsand the regulation of their production, several fundamental questions remain to be solved. These include questionsregarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity. Introduction Since many thousands of years mankind has exploitedthe fact that lactic acid bacterium (LAB) fermentationis an effective method for prolonged safe storage of foodstuff. The preservative effect of LAB is primarilydue to homolactic fermentation of sugar, which res-ults in the production of large amounts of lactic acid(Axelsson 1998). The resulting lowering of the pH(which may reach values lower than 4.0) effectivelyprevents outgrowth of almost all potential spoilagemicroorganisms.In the past century it has gradually become clearthat LAB combatmicroorganismsby at least oneothermechanism, namely by secretion of anti-microbialpeptides. These peptides are termed bacteriocins andshould not be confused with  protein -bacteriocinsfromGram-negative bacteria (e.g. colicins). Not surpris-ingly, LAB bacteriocins seem to be primarily aimed atother LAB, which are likely to be the most prominentcompetitor in the (acidic) ecological niche in whichthese bacteria reside. However, LAB bacteriocins alsoshow activity towards a number of potential Gram-positive food spoilage and/or pathogenic bacteria, forexample towards  Listeria .LAB are generallyregardedas safe and so are theirbacteriocins, which do not affect humans or other eu-karyotes. Bacteriocins are thus receiving lots of atten-tion, since applications as ‘natural’ food preservativesandmaybeevenasantibioticsmaybeenvisaged.Sinceabout 1990 the field has grown dramatically and thishas led to the discoveryand characterizationof a largenumber of bacteriocins.Concomitantly with the discovery of new bacteri-ocins, several interesting aspects of these peptideshave been revealed. These include regulation of pro-  640duction, structure-function relationships, as well asaspects of immunity (self-protection), target cell sens-itivity and resistance. Below, some recent develop-ments in the bacteriocin field are reviewed, with focuson the (potential) contribution of class II bacteriocinsto competition amongst LAB. Further details on sev-eral of the topics discussed here as well as overviewsof related topics that are not addressed in this pa-per (e.g. production of class I bacteriocins) may befound in several recent review papers (Nes et al. 1996;Montville & Chen 1998; McCafferty et al. 1999;Kleerebezem et al. 1999; Nes & Eijsink 1999; Enna-har et al. 2000; Nes & Holo 2000; Kleerebezem &Quadri 2001; McAuliffe et al. 2001). Bacteriocin diversity Bacteriocins are synthesized as precursors on the ri-bosome and the mature peptides usually consist of 20 – 60 amino acids. There is large variation amongthe peptides, e.g. in terms of length, amino acidsequence and composition, secretion and processingmachinery, post-translational modifications, and anti-microbial activity (alone or in combination with otherpeptides). Almost all bacteriocin peptides have a netpositive charge at neutral or slightly acidic pH andthey usually contain stretches of sequence that arehydrophobicand/or amphiphilic.Several attempts have been made to classifyLAB bacteriocins (Klaenhammer 1993; Nes et al.1996; Holo & Nes 2000, McAuliffe et al. 2001).The currently accepted classification is as follows:Class I: LantibioticsType A: Elongated shaped moleculesType B: Globular moleculesClass II: Nonmodified heat-stable bacteriocins(peptides)Subclass IIa: Pediocin-like bacteriocinsSubclass IIb: Two-peptide bacteriocinsSubclass IIc: Other peptide bacteriocinsClass III: Protein bacteriocinsIn previous definitions, class IIc contained bacteri-ocins secreted via the  sec -dependent pathway, but,today, the secretion mechanism is no longer used as adiscriminator in the classification of non-lantibiotics.Only very few potential class III bacteriocins havebeen described, which are not discussed in this review.Lantibiotics (Schnell et al. 1988; Sahl & Bierbaum1998,McAuliffeetal. 2001)areproducedasprecursorpeptides which undergo extensive post-translationalmodifications. They have been found in LAB and invarious other Gram-positive bacteria, e.g. staphylo-cocci. The mature peptides contain modified aminoacids such as 2,3-didehydroalanine,  D -alanine, and2,3-didehydrobutyrine, and as well as characteristiclanthionine rings that result from thioether formationbetween the side chains of cysteine and serine orthreonine. The best known lantibiotic is nisin (TypeA), which has a broad anti-microbial spectrum andwhich is used as food preservative today. Structure-functionrelationshipsin nisin havebeen unravelledbyNMR, site-directed mutagenesis and numerous stud-ies on the mode-of-action of wild type and mutantpeptides (see McAuliffe et al. 2001, and referencestherein). Recently, Breukink et al. (1999), identifieda membrane receptor for nisin, thus showing how themolecule may interact with the target cell. The pi-oneering work on lantibiotics is of great importancefor studies of class II bacteriocins which are the mainsubject of this review.Class IIa bacteriocins are characterized by the oc-currenceof a YGNGVXCXXXXCXV sequencemotif in their N-terminal half, including two cysteines thatform a disulfide bridge (Eijsink et al. 1998; Figure 1).Another shared characteristic of these bacteriocins istheir stronginhibitoryeffect on  Listeria . Class IIa bac-teriocins have been encountered in a great variety of LAB belonging to the genera  Lactobacillus, Entero-coccus, Pediococcus, Carnobacterium,  and  Leucon-ostoc.  They have also been found in the non-LAB  Bifidobacterium bifidum  (Yildirim et al. 1999),  Ba-cillus coagulans  (Le Marrec et al. 2000) and  Listeriainnocua  (Kalmokoff et al. 2001).Class IIbbacteriocinsarebacteriocinswhoseactiv-ity dependson the complementaryactivity of two pep-tides (Nissen-Meyer et al. 1992). In some cases oneor both of the individual peptides may be completelyinactive, as seems to be the case for, e.g. lactococ-cin G from  Lactococcus lactis  (Nissen-Meyer et al.1992). In other cases, one or each of the individualpeptides may display some activity, but clear syner-gistic affects on activity are observed upon combiningthe two (e.g. Anderssen et al. 1998). It is important tonote that one-peptide bacteriocins may display syner-gistic effects when applied in combination; the termtwo-peptide bacteriocins (class IIb) refers only to sets  641 Figure 1.  Sequences of class IIa bacteriocins (pediocin-like bacteriocins), aligned by their N-terminal halves. No attempts were made to alignthe C-terminal halves of the peptides; residue numbering is according to the sequence of pediocin PA-1; cysteine residues are printed in boldface. References: pediocin PA-1, Nieto-Lozano et al. 1992; Henderson et al. 1992; Marugg et al. 1992 [pediocin PA-1 is identical to pediocinAcH (Motlagh et al. 1992)]; enterocin A, Aymerich et al. 1996; divercin V41, Metivier et al. 1998 [divercin V41 is identical to bavaricin MN(Kaiser & Montville 1996)]; coagulin, Le Marrec et al. 2000; sakacin P, Tichaczek et al. 1992 and Hühne et al. 1996 [sakacin P is identicalto bavaricin A (Larsen et al. 1993)]; listeriocin 743A, Kalmokoff et al. 2001; curvacin A, Tichaczek et al. 1992 [curvacin A is identical tosakacin A (Axelsson & Holck 1995)]; piscicolin 126, Jack et al. 1996 [piscicolin 126 is identical to piscicocin V1a (Bhugaloo-Vial et al.1996)]; leucocin A, Hastings et al. 1991; mesentericin Y105, Fremaux et al. 1995; carnobacteriocin B2, Quadri et al. 1994; carnobacteriocinBM1, Quadri et al. 1994 [carnobacteriocin BM1 is identical to piscicocin V1b (Bhugaloo-Vial et al. 1996)]; bacteriocin 31, Tomita et al. 1996;enterocin P, Cintas et al. 1997; mundticin, Bennik et al. 1998. of peptides whose genes are in the same operon (seebelow). Two-peptide lantibiotics do also exist (Navar-atna et al. 1998; Ryan et al. 1999; Holo et al. 2001),but these bacteriocins have not yet been classified as aseparate ‘type’ of class I bacteriocins.Class IIc contains all non-lantibiotic bacteriocinsthat do not belongto class IIa and IIb. Class IIc repres-ents a rather diverse set of bacteriocins derived from avariety of LAB. Despite the fact that some class IIcbacteriocins display significant sequence similarities(e.g. Casaus et al. 1997; Franz et al. 1999), so far nosub-groups have been established.Recently, Cintas et al. (1998) identified a two-component unmodified bacteriocin in  Enterococcus faecium  L50 (Enterocins L50A and L50B). This bac-teriocin lacks several of the characteristics of class IIbacteriocins, whereas it has many of the characterist-ics of a small group of cytolytic peptides secreted bycertainstaphylococci(e.g. Donvitoet al. 1997). Cintaset al. concluded that enterocins L50A and L50B thusbelong to a new class of LAB bacteriocins. Bacteriocin structure and mode of action NMR structures of the lantibiotic nisin under vari-ous conditions show that this peptide is very flexiblein aqueous solutions, with conformational restrictionsprimarily being imposed by the lanthionine rings (Vande Ven 1991; Lian et al. 1992). Further structuringis induced in trifluoroethanol containing solutions aswell as upon interaction with micelles or membranes(Van den Hooven et al. 1993, 1996). When interactingwith micelles, nisin adopts an amphiphilic, rod-likestructure with a relativelyflexible ‘hingeregion’in themiddle.So far, all structural studies of one- or two-component class II bacteriocins have shown thatthe peptides are unstructured in aqueous solutions,whereas partly helical structures are adopted in thepresence of trifluoroethanol, micelles and liposomes(Fleury et al. 1996; Fregeau Gallagher et al. 1997;Montville & Chen 1998; Hauge et al. 1998a, 1999;Wang et al. 1999). The most precise information onthe structure of class II bacteriocins comes from thework of J.C. Vederas, M.E. Stiles and co-workerswho have resolved the three-dimensional structuresof the 37-residue class IIa bacteriocin leucocin A(Fregeau Gallagher et al. 1997) and of the 48-residueclass IIa bacteriocin carnobacteriocin B2 (Wang etal. 1999) (see Figure 1 for primary sequences). Thetwo structures are similar in the sense that both pep-tides contain an amphiphilic  α -helix spanning from  642residues 17/18 to residues 31/39 in leucocin A andcarnobacteriocin B2, respectively. Most remarkably,the N-terminal parts (which have highly conserved se-quences; Figure 1) displayed different structures. Incarnobacteriocin B2, whose structure was only stud-ied in 90% trifluoroethanol, the N-terminal part washighly disordered. In leucocin A, the N-terminal partformed a three-stranded antiparallel  β -sheet in 90%trifluoroethanol and in the presence of dodecylphos-phocholine micelles. It is possible that the structuresobserved in the N-terminal parts are artefacts causedby solvent conditions (Wang et al. 1999), thus leav-ing uncertainty with respect to the actual structure of these parts. On the basis of these and other, circulardichroism-based studies, it seems certain though thatclass II bacteriocins adopt a partly  α -helical structurein their active conformations.It should be noted that the literature contains quitea number of structure-predictions for class II bacteri-ocins that are simply based on analyses of (aligned)primarysequences. Such predictions should be treatedwith great caution since they are made using softwarethat is developed for and optimised with the help of the structures of soluble proteins. It seems rather inap-propriate to use such packages to predict the structureof   peptides , that only adopt structure when interactingwith an entity as complex (charge, hydrophobicity) asa membrane.Bacteriocins generally act by creating pores in themembrane of their target cells. This has deleteriouseffectssuch as dissipationofprotonmotiveforce, ATPdepletionandleakageofnutrientsandmetabolites.Al-though the formation of pores is a general feature, thesize, stability and conductivity of these pores differsconsiderablyfrom bacteriocinto bacteriocin(e.g. Chi-kindas et al. 1993; Bruno & Montville 1993; Abee1995; Driessen et al. 1995; Gonzalez et al. 1996;Marciset et al. 1997; Bennik et al. 1997; Montville& Chen 1998; Moll et al. 1996, 1999a; Herranz et al.2001a,b).To form a pore, bacteriocins have to interactwith the cytoplasmic membrane of target cells. Thisprocess is at least in part governed by electrostaticinteractions between the positively charged peptideand anionic lipids that are abundantly present in themembranes of Gram-positive bacteria. For example,Chen et al. (1997) showed that different fragments of pediocin PA-1 have different affinities for anionic ves-icles and that this affinity is much more dominated bythe presence of positive charge than by the YGNGVconsensus sequence motif. Hauge et al (1998a, 1999)used circular dichroism to show that class IIb peptidesbecome much more structured upon interaction withanionic vesicles than with zwitterionic vesicles. Bind-ing of bacteriocins to the membrane and subsequentpore formation are usually affected by factors suchas the membrane potential in the target cell and thepH. A membranepotentialsupposedlyenhancesinser-tion of the bound bacteriocin into the membrane andmay also affect association of bacteriocin molecules.Consequently, bacteriocin sensitivity depends to someextent on the physiological state of the cell.One major question in the bacteriocin field con-cerns the question whether or not bacteriocins actthrough receptors in the target cell membrane. Arelated question concerns the character (protein-aceous?) and specificity of possible receptors. Nisinwas generally thought not to act via a specific re-ceptor because it creates functional pores in mem-branes that do not contain any proteins. It has re-cently been shown, however, that the peptidoglycanprecursor lipid II (undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc) specifically inter-acts with nisin and that this interaction facilitatespore formation quite substantially (Brötz et al. 1998;Breukink et al. 1999).The presence and character of receptor-moleculesfor class II bacteriocins is an unresolved issue. Inmany mode-of action studies, the bacteriocins testedwere only active towards whole cells or vesicles con-taining cellular proteins, but not towards protein-freevesicles. This obviously suggests the presence of areceptor in the cell membrane. However, conflictingdata exist as reviewed recently by Montville & Chen(1998), Moll et al. (1999b) and Nes & Holo (2000).Most importantly, Yan et al. (2000) recently showedthat the  D -enantiomerof leucocinA has no bacteriocinactivity, whichstronglysuggests that activityis relatedto a stereospecific interaction with a chiral target mo-lecule. There is evidence in the literature showing thatthesechiraltargetmolecules(‘receptors’)arecompon-ents of sugar phosphotransferasesystems (Ramnath etal. 2000; Héchard et al. 2001; Dalet et al., 2001). Per-haps, the situation for class II bacteriocins will turnout to be analogous to the situation for nisin, namelythat bacteriocin action is enhanced by interaction witha receptor molecule. The extent of such enhancementas well as the character of the receptor molecule mayturn out to vary among class II bacteriocins.Bacteriocins have often been proposed to formpores by a so-called ‘barrel-stave’ mechanism (Oj-cius & Young 1991). In this mechanism bacteriocinmolecules (that bind as monomers) adopt a trans-  643membrane localization and they associate to form atrans-membrane barrel with a hydrophilic interior anda hydrophobic exterior. This attractive mechanismmay be valid in some cases, but it is probably toosimple. Recent structuralstudies (see above)as well asmode of action studies indicate that the situation gen-erally is more complex. Recent models, including the‘wedge’ and the ‘carpet’ model, have been reviewedby Moll et al. (1999b).A more detailed description of structure-functionrelationships in bacteriocins as well as their possiblemodes of action is beyond the scope of this review.Such issues have been addressed recently in reviewsby e.g. Montville & Chen (1998), Breukink & DeKruijff (1998), Moll et al. (1999b) and Ennahar etal. (2000). As yet, the published amount of sys-tematic (site-directed mutagenesis-based) structure-function studies of class II bacteriocins is rather lim-ited (Fleury et al. 1996; Quadri et al. 1997a; Fimlandet al. 1996, 1998; Miller et al. 1998). Recently, how-ever, a new expression system for class II bacteriocinshas been developed (Axelsson et al. 1998) which isnow used to produce large numbers of pure mutants(Fimland et al. 2000; Johnsen et al. 2000; Fimlandet al., 2002). Characterization of these mutants willprovide new insight into how bacteriocins work. Resistance and immunity Several factors may contribute to making a cell resist-ant towards bacteriocins. The composition and struc-ture of both cell wall and cellular membrane(s) maybesuch that the bacteriocin is physically unable to reachits target. Alternatively, as referred to above, certaincellular components (‘receptors’) that are essentialfor or augment bacteriocin action may be lacking ormutated. One might also speculatethat the presence of (aspecific) proteases in and near the target cell may re-duce bacteriocin effectiveness in some cases. Finally,the physiological state of the target cell may affect theease at which a membrane-bound bacteriocin actuallycan form pores.Insensitivity towards bacteriocins may also be ob-tained in a more specific manner, namely via theexpression of dedicated immunity genes. Bacteriocinproducing LAB are insensitive to their own bacteri-ocins because they express cognate immunity genes,which are often co-transcribed with the structuralgene(s) for the bacteriocin(s) (Nes et al. 1996). Bothsequence analysis and experimentalwork indicate thatimmunity proteins for class II bacteriocins are solubleand mainly located in the cytoplasm, althoughin somecases the presence of one transmembrane helix hasbeen suggested (Nissen-Meyer et al. 1993; Quadri etal. 1995; Venema et al. 1995; Dayem et al. 1996;Eijsink et al. 1998).Very little is known about the mode of action of these immunity proteins and there does not exist a re-liable model for immunity protein action (Nes & Holo2000; Ennahar et al. 2000). For class II bacteriocins,most experimentalwork has so far been done on small(100–150 residues) immunity proteins belonging toone-peptide bacteriocins. Van Belkum et al. (1991)andChikindasetal. (1993)showedthatwhereasmem-brane vesicles from sensitive cells became leaky uponbacteriocin exposure, vesicles from immune cells didnot. Venema et al. (1995) showed that the C-terminalpart of the lactococcin A immunity protein is exposedto the exterior of the cell. Although this observationsuggests that direct interactions between the immunityprotein and the bacteriocin are possible, most res-ults in the literature indicate that such interactions donot occur. For example, there are no indications thatbacteriocins actually bind to immunity proteins (e.g.Quadri et al, 1995). Furthermore, it has been shownthat sensitive cells are not protected from bacteriocinaction by adding immunity protein to the culture me-dium (Nissen-Meyer et al. 1993; Quadri et al. 1995).This may be taken to indicate that immunity proteinsact via an effect on a (putative) bacteriocin receptor inthe cytoplasmic membrane (e.g. Venema et al. 1995).The immunity proteins of one-peptide class II bac-teriocins are not completely specific, as illustrated byseveral studies showing that one immunity gene cangive protection towards several bacteriocins (Eijsink et al. 1998; Franz et al. 2000; Fimland G, Eijsink-VGH & Nissen-Meyer J, unpublished observations).The cross-immunity properties of immunity proteinsare difficult to rationalize since these proteins displayremarkably high sequence variation that is not or to avery little extend correlated with variation in the se-quences of their cognate bacteriocins. For example,sequence identities between pairs of immunity pro-teins of class IIa bacteriocins are most often below30 percent, despite considerable sequence similartiesbetweenthebacteriocinsthemselves(Figure1; Eijsink et al. 1998).Interestingly, many bacteriocin producing LABcontain more (putative) immunity genes than wouldseem necessary for protection against their own bac-teriocins (see Brurberg et al. 1997 for an example).
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