Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/23796686

Structure-FunctionRelationshipsoftheNon-Lanthionine-ContainingPeptide(classII)BacteriocinsProducedbyGram-PositiveBacteria

ARTICLEinCURRENTPHARMACEUTICALBIOTECHNOLOGY·FEBRUARY2009

ImpactFactor:2.51·DOI:10.2174/138920109787048661·Source:PubMed

CITATIONS

102

READS

160

5AUTHORS,INCLUDING:

CamillaOppegård

UniversityofOslo

12PUBLICATIONS293CITATIONS

SEEPROFILE

HelénSophieHaugen

UniversityofOslo

6PUBLICATIONS198CITATIONS

SEEPROFILE

PerEugenKristiansen

UniversityofOslo

35PUBLICATIONS811CITATIONS

SEEPROFILE

Availablefrom:HåvardJHaugen

Retrievedon:04February2016

Current Pharmaceutical Biotechnology, 2009, 10, 19-37 19

1389-2010/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Structure-Function Relationships of the Non-Lanthionine-Containing Pep-tide (class II) Bacteriocins Produced by Gram-Positive Bacteria

J. Nissen-Meyer*, P. Rogne, C. Oppegård, H.S. Haugen and P.E. Kristiansen

Department of Molecular Biosciences, University of Oslo, Oslo, Norway

Abstract: This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacterio-

cins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial

one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacterio-

cins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like one-

peptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through mem-

brane-permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with

target membranes, their cationic N-terminal half forms a -sheet-like structure that binds to the target cell surface,

while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell

membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane

leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacterio-

cin-permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide

bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacterio-

cins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacte-

riocins kill target-cells through basically the same mechanism; the common theme being that a membrane-

penetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby

causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

Keywords: Bacteriocins, antimicrobial peptides, immunity proteins, pediocin-like bacteriocins, two-peptide bacteriocins, cy-clic bacteriocins.

INTRODUCTION

Ribosomally synthesized antimicrobial peptides (AMPs) are widely distributed in nature; they are produced by bacte-ria, plants and a wide variety of animals - both invertebrates and vertebrates [1-6]. For animals and plants, AMPs are an important defense against microorganisms. AMPs may also for bacteria be thought of as a type of defense, since AMPs enable killing of invading bacteria that compete with the AMP-producer for nutrients. Eukaryotic and bacterial AMPs superficially resemble each other in that they are often mem-brane permeabilizing, cationic, and amphiphilic or hydro-phobic [2]. The bacterial AMPs - commonly referred to as bacteriocins - are, however, generally much more potent than the eukaryotic AMPs; the former being active at nanomolar concentrations and the latter at micromolar concentrations. Structure-function analysis of bacterial AMPs is therefore particularly relevant for illuminating how highly potent membrane permeabilizing AMPs function at a molecular level.

There has especially been great interest in structure-function analysis of AMPs/bacteriocins produced by lactic acid bacteria (LAB) because of their “food grade quality” and industrial importance. LAB are used in food and feed

*Address correspondence to this author at the Department of Molecular

Biosciences, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Nor-

way; Tel: +47-22 85 73 51; Fax: +47-22 85 44 43; E-mail: jon.nissen-meyer@imbv.uio.no

production, they are part of the natural microbial flora in food humans have consumed for centuries, and they consti-tute a significant part of the indigenous flora of mammals, including humans. LAB and LAB bacteriocins may, conse-quently, be considered to be relatively safe agents for pre-venting growth of pathogenic/undesirable micro-organisms. The LAB bacteriocins nisin and pediocin PA-1 are in fact presently used as food preservatives [6], and the potential of LAB bacteriocins in medical applications may be exempli-fied by recent results showing that oral intake of bacteriocin-producing LAB protects mice against lethal doses of Listeria monocytogenes [7]. Optimal and rational exploitation of bac-teriocins as antimicrobial agents requires, however, insight into how bacteriocins function at a molecular level, and this in turn necessitates insight into their three-dimensional struc-tures, as bacteriocins function through structural interactions. We present here an overview of structure-function studies on non-lanthionine-containing LAB bacteriocins, studies that have given us insight at a molecular level into structural fea-tures that are important for the biosynthesis, mode of action, and potency of bacteriocins. Knowledge gained from these studies may allow future development of bacteriocins into drugs for treatment of infections and into additives for pres-ervation of food and animal feed. The development of bacte-riocins into new antimicrobial agents is clearly of consider-able importance in view of the dramatic increase in antibi-otic-resistant pathogenic bacteria and the undesirable side effects that many chemical preservatives may have. Bacte-riocins exert their antimicrobial activity in a different manner

20 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

than antibiotics and preservatives, and may therefore be able to complement or possibly substitute antibiotics and pre-servatives.

TWO MAIN CLASSES OF LAB BACTERIOCINS: THE LANTHIONINE-CONTAINING (CLASS-I) BAC-TERIOCINS AND THE NON-LANTHIONINE-CONT-

AINING (CLASS-II) BACTERIOCINS

The LAB peptide bacteriocins may be divided into two main classes. Class-I consists of the lanthionine-containing post-translationally modified bacteriocins, commonly re-ferred to as lantibiotics due to the presence lanthionine [5, 6]. Lanthionine may be described as a D-alanine (derived from a serine residue) and an L-alanine (derived from a cysteine residue) linked by a sulphur atom that forms a thioether bond that presumably stabilizes three-dimensional structures in an analogous manner as the more common disulfide bond. Class-I bacteriocins also often contain methyl-lanthionine, and other non-standard residues such as dehydroalanine, dehydrobutyrine and D-alanine [5, 6, 8]. Class-II consists of the non-lanthionine-containing peptide bacteriocins that are not subject to extensive post-translational modifications [6]. Class-II bacteriocins may be divided into four sub-classes, class-IIa, -IIb -IIc, and -IId [6]. Class-IIa consists of the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences (reviewed in [9, 10]). Class-IIb contains the two-peptide bacteriocins (reviewed in [11, 12]). These bacteriocins are unique in that they consist of two very different peptides and optimal antimicrobial activity requires the presence of both peptides in about equal amounts [11, 12]. Class-IIc consists of the cyclic bacteriocins whose N- and C-termini are covalently linked, resulting in a cyclic structure [6]. The linear non-pediocin-like one-peptide bacte-riocins that show no sequence similarity to the pediocin-like bacteriocins are placed in class-IId [6]

1. This review focuses

on the non-lanthionine-containing (class-II) bacteriocins, since structure-function aspects of the lanthionine-containing class-I bacteriocins are discussed elsewhere in this volume. Moreover, the review deals mainly with LAB bacteriocins, as they are the ones that are overall best characterized, but non-LAB non-lanthionine-containing peptide bacteriocins will also be discussed to the extent that their structures have been analysed.

THE PEDIOCIN-LIKE (CLASS-IIA) BACTERIOCINS

The pediocin-like (class-IIa) bacteriocins are produced by a variety of lactic acid bacteria and constitute perhaps the most important and well-studied group of class-II bacterio-cins (reviewed in [9, 10]). There has been considerable inter-est in these bacteriocins, not the least because of their anti-listerial activity [13, 14] combined with the fact that they are produced by “food grade” bacteria. Since the discovery and characterization of leucocin A [15], sakacin P [16-18], cur-vacin A [16, 19-21], mesentericin Y105 [22-24], and pedio-cin PA-1 [25-28] (from which the term pediocin-like bacte-riocins/AMPs has been derived) in the early nineties, the group has expanded and includes now more than 20 bacte-riocins [15-53] (Fig. (1)). They are all cationic and partly

1 This classification is according to that proposed by Cotter et al. [6]. In earlier classifi-

cation schemes the class-IId bacteriocins were placed in class-IIc or included in class-

IIa along with the pediocin-like bacteriocins.

amphiphilic and/or hydrophobic, have between 37 and 48 residues, and in the N-terminal region they all contain the conserved Y-G-N-G-V/L “pediocin box” motif and two cys-teine residues joined by a disulfide bridge (Fig. (1)). Based on their primary structures (Fig. (1)), their peptide chains may be divided into two regions: (i) a cationic, hydrophilic and highly conserved N-terminal region (up to about residue 17) that contains the “pediocin box” motif, and (ii) a less conserved more hydrophobic C-terminal region (from about residue 18) [10, 54]. They may be grouped into three or four subgroups according to sequence similarities and differences in the less conserved C-terminal region (Fig. (1)) [10]. The peptides in subgroup-1, -3 and -4 are clearly somewhat longer – because of a longer C-terminal region – than the peptides of subgroup-2. The peptides in subgroup-3 (and enterocin SE-K4 and carnobacteriocin B2 in subgroup-4) lack the hairpin-stabilizing tryptophan and/or cysteine resi-dues that are present at or near the C-terminal end in all pep-tides of subgroup-1 and -2 and in most of the peptides in subgroup-4 (Fig. (1)).

The Pediocin-Like Bacteriocins Permeabilize Target-Cell Membranes

The pediocin-like bacteriocins are active - but with dif-ferent potencies – against various Lactobacillus, Lactococ-cus, Enterococcus, Pediococcus, Leuconostoc, Carnobacte-rium, Clostridium, and Listeria strains [13, 14]. The bacte-riocins kill the cells by permeabilizing the cell membrane, resulting in disruption of the cell’s proton motive force [55-63] and depletion of the ATP pool, probably due to ATP consumption connected to the cell’s attempt to restore the proton motif force. The bacteriocins’ positive charge appar-ently facilitates the initial interactions with the negatively charged bacterial phospholipid-containing membranes and or acidic bacterial cell walls, whereas their amphiphilic/ hydro-phobic character enables membrane-permeabilization. Inter-estingly, despite similarities in primary structures, the pedio-cin-like bacteriocins differ markedly in their ability to kill different target-cells [13, 64-66]. This difference in their tar-get-cell specificity combined with their extensive sequence similarities make these bacteriocins especially well suited for analyzing the relationship between structure and target-cell specificity.

The Three-Dimensional Structure of Pediocin-Like Bac-teriocins

Circular dichroism (CD) spectroscopy and nuclear mag-netic resonance (NMR) structural analysis of the five pedio-cin-like bacteriocins leucocin A [67], carnobacteriocin B2 [68], curvacin A [69], sakacin P [70], and a mutant of sa-kacin P [70] revealed that the pediocin-like bacteriocins are unstructured in aqueous solution, but become structured upon contact with membrane-mimicking entities [67, 69-71]. The conserved N-terminal region (up to about residue 17) forms then a three-stranded anti-parallel -sheet-like struc-ture that is stabilized by a conserved disulfide bridge, while the more hydrophobic C-terminal region forms – with a few exceptions (see below) – a hairpin-like structure that consists of an amphiphilic -helix (from about residue 18 to about residue 33) followed by a rather extended C-terminal tail that folds back onto the central -helix (Fig. (2)) [67, 70]. There

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 21

is a flexible hinge (at the conserved Asp-17 in subgroup-1 bacteriocins; Fig. (1)) between the -sheet N-terminal region and the hairpin-like C-terminal region and one thereby ob-tains two domains that may move relative to each other [70].

The hairpin structure is in some pediocin-like bacterio-cins (such as enterocin A, divergicin M35, divercin V41, coagulin, pediocin PA-1, sakacin G and plantaricin 423 (Fig. (1))) stabilized by a disulfide bridge between a cysteine resi-due in the middle of the -helix and a cysteine residue at the C-terminus (Fig. (2A)). However, most pediocin-like bacte-riocins lack these two cysteine residues, but instead contain a well-conserved central tryptophan residue (at position 18 in most of the peptides (Fig. (1)) and a tryptophan residue near the C-terminus (exceptions being bacteriocins that belong to subgroup-3, and enterocin SE-K4 and carnobacteriocin B2 in subgroup-4; Fig. (1)). Site directed in vitro mutagenesis has revealed that these two tryptophan residues position them-selves in the membrane-water interface and thereby stabilize the hairpin structure of the peptides that lack the structure-stabilizing disulfide bridge in the C-terminal domain [66] (Fig. (2B); discussed in more detail in the next section deal-ing with orientation in membranes). Consistent with this hairpin structure model is the observation that insertion of a hairpin-stabilizing disulfide bridge into a pediocin-like bac-teriocin (sakacin P) that lacks this bridge does not have a detrimental effect on the activity of the peptide, but rather rendered it more thermostable. The sakacin P variant with a hairpin-stabilizing disulfide bridge thus functions at higher temperatures; it has the same potency at 20 and 37

oC,

whereas wild-type sakacin P is approximately 10 times less potent at 37 than at 20

oC [64]. Also consistent with the hair-

pin structure model is the observation that the deleterious effect of replacing the central tryptophan residue (i.e. Trp-18 in most of the peptides; Fig. (1)) or the tryptophan residue near the C-terminus with a hydrophobic residue is largely overcome upon also introducing a hairpin-stabilizing disul-fide bridge (between residues 24 and 44) in the C-terminal domain [66].

It should be noted at this point that the amino acid se-quences of the C-terminal domain of the pediocin-like bacte-riocins in subgroup-3 (and enterocin SE-K4 and carnobacte-riocin B2 in subgroup-4) differ conceptually from the corre-sponding sequences of the bacteriocins of subgroup-1 and -2, in that the former lack both the hairpin-stabilizing disulfide bridge and the well conserved hairpin-stabilizing tryptophan residue near the C-terminal end (Fig. (1)). It is, consequently, not entirely clear whether or not the bacteriocins in sub-group-3 (and enterocin SE-K4 and carnobacteriocin B2 in subgroup-4) have a hairpin-like structure in their C-terminal domain. The determination of the three-dimensional struc-ture of the bacteriocin curvacin A (belongs to subgroup-3) revealed that its C-terminal half forms a helix-hinge-helix structure that clearly differs from the hairpin-like structure that is typical for the C-terminal domain of the pediocin-like bacteriocins that belong to subgroup-1 and -2 [69]. The two structures (i.e. the hairpin-like and the helix-hinge-helix structures) may, however, be functionally equivalent, as both structures constitute the part of pediocin-like bacteriocins that penetrates into the hydrophobic core of target mem-branes. Moreover, the target-cell specificity of hybrid bacte-riocins in which N- and C-terminal domains from different

pediocin-like bacteriocins have been joined together indi-cates that the C-terminal domain of all pediocin-like bacte-riocins is the major target-cell specificity determinant and the part that is specifically recognized by immunity proteins. Immunity proteins protect bacteriocin-producing bacteria from being killed by their own bacteriocin (see section below on target-cell specificity). Analysis of genetically modified variants of curvacin A in a similar manner as has been done for other pediocin-like bacteriocins (see next section on ori-entation in membranes) should reveal how the helix-hinge-helix structure in curvacin A (and possibly a few other pediocin-like bacteriocins, such as enterocin P and carnobac-teriocin BM1) orients in target-cell membranes.

Orientation of the Pediocin-Like Bacteriocins in Mem-branes

Site directed in vitro mutagenesis and peptide-binding studies indicate that the cationic N-terminal -sheet-like do-main mediates binding of the pediocin-like bacteriocins to the target cell surface through electrostatic interactions [72, 73]. Thus, bacteriocin variants in which the net positive charge in the N-terminal -sheet-like domain is reduced by one by substituting a positively charged residue with a neu-tral polar residue exhibit reduced binding to target cells and a 2-100 fold reduction in activity [73-75]. Moreover, substitut-ing residues in the well-conserved (Y)-Y-G-N-G-V/L “pediocin box” motif in the N-terminal domain affect the antimicrobial activity and bacteriocin-liposome interactions in a manner that suggests that the N-terminal -sheet-like domain positions itself in the membrane interface region [76]. This would enable the more hydrophobic C-terminal hairpin-like domain to penetrate obliquely into the hydro-phobic core of target membranes, the hinge providing the structural flexibility that would permit the domain to dip into the membrane core and thereby mediate membrane-leakage.

Several studies involving modified pediocin-like bacte-riocins reveal that the hydrophobic C-terminal hairpin-like domain is in fact the part of these bacteriocins that penetrates into the hydrophobic core of target membranes. In one of these studies [77], the DNA sequence encoding pediocin PA-1 was fused to the sequence encoding the 43 kDa maltose-binding protein, thereby joining the N-terminal end of pedio-cin PA-1 with the C-terminal end of the maltose-binding protein. Interestingly, this chimeric protein had antimicrobial activity, suggesting that the N-terminal -sheet-like domain remains on the cell’s outside and that the C-terminal hairpin domain must, consequently, be the part that penetrates into the core of target membranes [77]. In another study [66], the effect of mutating each of three highly conserved tryptophan residues (i.e. Trp-18, at the beginning of the helix; Trp-33, near the end of the helix; Trp-41 near the C-terminus) in the C-terminal hairpin-like domain of sakacin P was studied. Replacement of Trp-18 and -41 with other aromatic residues was much less detrimental than replacement with a hydro-phobic or hydrophilic residue [66]. In contrast, replacement of Trp-33 with hydrophobic residues did not have a negative effect on the activity, but replacement with aromatic or polar residues was detrimental. Taken together, the results indi-cated that both Trp-18 and -41 position themselves close to each other in the water-membrane interface (as is common for aromatic residues [78]; Fig. (2B)). The open-ended side

22 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

Fig. (1). Multiple sequence alignment of pediocin-like bacteriocins. The C-terminal half is more diverse than the N-terminal half, and the

classification of the peptides into four subgroups is based on sequence similarities and differences in the C-terminal half. There is a flexible

hinge at the conserved Asp17 (in green) in peptides of subgroup-1, and presumably also at Asn17/Asp17 (in green) in peptides of subgroup-2,

-3 and -4. This hinge separates the -sheet N-terminal domain and the more diverse C-terminal domain. The residues are colored as follows:

Asn, Asp, Gln and Glu are in green; Ile, Leu, and Val are in purple; Trp, Tyr and Phe are in black; Cys in grey; Ala and Gly in yellow;

Arg, Lys and His in blue; Ser and Thr in red; Pro in white; Met in dark grey. Note that in numbering the residues (as indicated above

the sequences), residue number 2 before the well conserved YGNGV motif is in all cases referred to as residue 1, since this residue is

the first residue in most - but not all - of the peptides.

Fig. (2). A cartoon depiction of the structure and orientation in membranes of pediocin-like bacteriocins that belong to subgroup-1

and -2. A pediocin-like bacteriocin in which the C-terminal hairpin structure is stabilized by (A) a disulfide bridge (such as in enterocin A,

divercin V41, divergicin M35, coagulin, pediocin PA-1, sakacin G, and plantaricin 423), and by (B) an interface-localized tryptophan residue

near the C-terminal end of the bacteriocin (such as in the other subgroup-1 and -2 bacteriocins). Tryptophan residues that become localized in

the membrane-water interface are indicated by a red or blue W, tryptophan residues that become localized in the hydrophobic core of mem-

brane by a black W, and the disulfide bridge by -S-S-.

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 23

of the hairpin structure becomes thereby positioned in the membrane interface, while the other side of the hairpin struc-ture, near Trp-33 where the peptide backbone makes a turn and folds back on itself, penetrates more deeply into the hydrophobic core of the membrane (Fig. (2A)). The hairpin-like structure is thus stabilized and oriented correctly in the membrane. Consistent with this model is a more recent study in which the orientation of mesentericin Y105 in membranes was analyzed [74]. Tryptophan-fluorescence was measured upon adding to lipid micelles different variants of mesen-tericin Y105 that have only one tryptophan residue (not two as in the wild-type peptide) positioned either in the C-terminal end (position 37) or near the middle of the bacterio-cin (position 18). The fluorescence emission data revealed that both of these tryptophan residues positioned themselves near the membrane surface, presumably in the membrane interface, consistent with the hairpin-like structure model described above.

The Membrane-Penetrating C-terminal Domain is an Important Target-Cell Specificity Determinant

The structure of the pediocin-like bacteriocins suggests that the N- and C-terminal domains may to some extent function independently of each other. This is consistent with studies showing that hybrid bacteriocins created by joining N- and C-terminal domains from different pediocin-like bac-teriocins (using the hinge between the domains as the re-combination point) were often as active as the parental bacte-riocins [79]. These hybrid bacteriocins had target-cell specificities similar to the bacteriocin from which the C-terminal domain was derived, indicating that the membrane-penetrating C-terminal domain is the major specificity de-terminant in pediocin-like bacteriocins [79]. This is consis-tent with studies showing that pediocin-like bacteriocins that have been altered in the C-terminal domain by in vitro mutagenesis often differ from the parental bacteriocin in their target-cell specificity [64, 66]. An important specificity-determining step therefore appears to involve interactions with lipids and/or proteins in the hydrophobic part of the target-cell membrane [79]. One protein that appears to be involved in such an interaction is the membrane-associated mannose phosphotransferase system permease [80-86]. This protein must be present for cells to be sensitive to pediocin-like bacteriocins [80, 82-86]. Comparative two-dimensional gel studies revealed that leucocin A-resistant cells derived from leucocin A-sensitive listerial strains lacked the MptA subunit of this protein [83]. Moreover, heterologous expres-sion of the MptC subunit of the mannose phosphotransferase permease in an insensitive strain of Lactococcus lactis ren-dered the strain sensitive to several pediocin-like bacterio-cins [85]. A more recent study showed that pediocin-like bacteriocins in fact bind to a part of the mannose phos-photransferase permease (the MptC and/or MptD subunit) that is embedded in the cell membrane [87]. Moreover, the study revealed that immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind strongly to the bacteriocin-permease complex and thereby prevent bac-teriocin-induced killing (see next section). Interactions be-tween pediocin-like bacteriocins and the mannose phos-photransferase permease thus apparently alter the conforma-tion of the permease in a manner that results in membrane-

leakage, and this leakage may be blocked on the cytosolic side of the membrane by the binding of an immunity protein to the bacteriocin-permease complex. Interestingly, 15-mer fragments covering most of the helical region of the pedio-cin-like bacteriocins inhibit the bacteriocins in a specific manner [88-90], suggesting that the helical part of the C-terminal domain might engage in a chiral helix-helix interac-tion with the mannose phosphotransferase system permease in the hydrophobic core of target membranes. This is consis-tent with activity measurements and CD secondary structural analysis carried out at different temperatures with several pediocin-like bacteriocins [91]. The study suggests that the

-helical region of these bacteriocins is important for target recognition and activity.

The Membrane-Penetrating C-terminal Domain of Pediocin-Like Bacteriocins Recognizes the C-Terminal

Part of Immunity Proteins

Bacteria that produce pediocin-like bacteriocins also pro-duce cognate immunity proteins that protect the bacteria from being killed by their own bacteriocins [18, 21, 79, 92-97]. The function of immunity proteins was demonstrated by showing that heterologous expression of their genes in sensi-tive bacteria rendered the bacteria less sensitive to pediocin-like bacteriocins. The amino acid sequences of at least 20 immunity proteins for pediocin-like bacteriocins have pres-ently been deduced from DNA sequences (reviewed in [10]). They display 5 to 85% sequence similarities, contain be-tween 88 and 115 amino acid residues, and they show a high degree of specificity in that they confer resistance only to their cognate pediocin-like bacteriocin and in some cases to a few bacteriocins that are closely related to the cognate bacte-riocin [79, 96, 97]. The immunity protein gene is often on the same operon as the gene encoding the cognate pediocin-like bacteriocin, and expression of the two genes is therefore often co-regulated [13]. The NMR solution and crystal struc-tures of the immunity proteins for three pediocin-like bacteriocins (carnobacteriocin B2, enterocin A and pediocin PA-1) have revealed that the proteins consist of an anti-parallel four-helix bundle with a C-terminal region that appears to be structurally somewhat flexible [98-101]. Structural modeling on the basis of sequence similarities suggests that other immunity proteins for pediocin-like bacteriocins may have similar structures [100].

The immunity proteins for pediocin-like bacteriocins are located intracellularly. Although these immunity proteins are relatively hydrophilic, some of the immunity proteins (about 1%) may be loosely associated with the internal side of the cell membrane [94, 95]. The immunity proteins are in fact expected to act via association with the internal side of the cell membrane, since it has been demonstrated using hybrid pediocin-like bacteriocins and hybrid immunity proteins that the membrane-penetrating C-terminal bacteriocin domain specifically interacts with the C-terminal half of the immu-nity proteins, perhaps via the structurally somewhat flexible C-terminal tail of the immunity proteins [79]. It has, how-ever, not been possible to demonstrate direct physical inter-action between immunity proteins and bacteriocins [98]. In view of the facts that (i) there is a strain-dependent variation in immunity protein functionality [96, 97] and that (ii) the membrane-permeabilizing C-terminal bacteriocin domain is

24 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

not likely to penetrate completely through the bacteria mem-brane, it has been suggested that the interaction is indirect [10, 79, 98]. It was suggested that the indirect interaction was via binding of the immunity protein to the cytoplasmic side of the bacteriocin receptor (i.e. the mannose phos-photransferase permease) and that this binding blocks the receptor’s ability to interact with the bacteriocin in a manner that leads to membrane-leakage [10, 79, 98]. As mentioned above, the recent study by Diep et al. [87] indeed reveals that the immunity proteins bind strongly to the mannose phos-photransferase permease, but only if the bacteriocin is also bound to the permease. The immunity proteins must there-fore recognize a bacteriocin-induced structural alteration in the permease, which results in binding of an immunity pro-tein to the permease-bacteriocin complex and blockage of bacteriocin-induced membrane-leakage. This mechanism of action necessitates, however, that the strain-dependent varia-tion in the receptor (mannose phosphotransferase permease) is large enough to cause sufficient variations in the receptor-binding of (i) the various pediocin-like bacteriocins (in order to account for the different target cell specificities of the pediocin-like bacteriocins), and (ii) the various immunity proteins (in order to account for specificity of immunity pro-teins for their cognate bacteriocins).

Involvement of Leader Sequence in Export of Pediocin-Like Bacteriocins

Pediocin-like bacteriocins (and most other class II bacte-riocins) are typically synthesized as pre-bacteriocins that contain a 15 to 30 residue-long N-terminal leader sequence with a consensus motif. The leader sequence, which is thought to form an -helix [102], is often of the double-glycine type that is cleaved off at the C-terminal side of two glycine residues [4, 103, 104], although three pediocin-like bacteriocins (enterocin P, bacteriocin 31 and listeriocin 743A) have instead a sec-type leader sequence. These three bacteriocins are apparently secreted by the sec-dependent translocation system [34, 46, 47], whereas the bacteriocins with a double-glycine leader sequence are secreted by a dedicated membrane-associated ABC transporter that con-comitantly cleaves off the leader sequence. The role that the leader sequence plays in targeting class-II bacteriocins to their dedicated ABC-transporters was determined by fusing the double-glycine leader of the pediocine-like bacteriocin leucocin A and the class-IId bacteriocin lactococcin A to divergicin A, a non-related class-IId bacteriocin that is nor-mally secreted by the cell’s general secretion pathway rather than via ABC-transporters [105]. The results showed that the double-glycine leader indeed functions as a signal for target-ing bacteriocins to their cognate transporter system, since divergicin A with a fused double-glycine leader from either lactococcin A or leucocin A was secreted by both the dedi-cated lactococcin A and leucocin A ABC-transporters [105]. Moreover, by substituting several residues in the 24-residue double-glycine leader of the pediocin-like bacteriocin mes-entericin Y105, several hydrophobic residues and the two glycine residues at position -1 and -2 were shown to be re-quired for optimal secretion of the bacteriocin and removal of the leader [106]. The glycine residues at position –1 and –2 appear to be especially important for correct cleavage of the double glycine-type leader sequence present in pre-mesentericin Y105. The double-gycine leader thus deter-

mines to a large extent whether or not the bacteriocin pre-form is recognized and exported by the dedicated ABC-bacteriocin-transporters [105]. It has been suggested that the leader sequence may also function to keep the bacteriocin inactive until it has been secreted from cells [102], consistent with results showing that the double-glycine leader inacti-vates the lantibiotic nisin [107]. The pre-form of pediocin PA-1 has, however, been reported to be active [108].

THE TWO-PEPTIDE (CLASS-IIB) BACTERIOCINS

The two-peptide (class-IIb) bacteriocins are unique in that they consist of two very different peptides and optimal activity requires both peptides in about equal amounts [2, 4, 11, 12]. At least 15 two-peptide bacteriocins have been iso-lated and characterized [109-132] (Fig. (3A and B)) since the first identification of such a bacteriocin (lactococcin G) in 1992 [109].

The Two-Peptide Bacteriocins Permeabilize Target-Cell Membranes

All two-peptide bacteriocins whose mode of action has been studied, this includes thermophilin 13 [122], lactococ-cin G [133, 134], plantaricin E/F [135], plantaricin J/K [135], lactacin F [136], and lactocin 705 [137], render target-cell membranes permeable to a variety of small molecules. These two-peptide bacteriocins appear to display some specificity with respect to which small molecules they conduct across membranes, and the various bacteriocins seem to differ somewhat in their specificities. For instance, lactococcin G permeabilizes target-cell membranes for a variety of mono-valent cations, such as Na

+, K

+, Li

+, Cs

+, Rb

+ and choline,

but not for divalent cations (such as Mg++

) or anions (such as phosphate), nor for H

+ [133, 134]. Plantaricin E/F and plan-

taricin J/K also permeabilize membranes for monovalent cations, including H

+ (in contrast to lactococcin G), but not

for divalent cations (Mg++

) or anions (phosphate) [135]. It appears, however, as if plantaricin E/F conducts cations more efficiently than plantaricin J/K and vice versa for ani-ons [135]. Lactacin F renders membranes permeable to K

+

and phosphate [136], and thermophilin 13 dissipates both the trans-membrane electrical potential and pH gradient, but its specificity with respect to molecules it conducts across membranes is not characterised [122]. Bacteriocin-induced leakage of various ions clearly dissipates the trans-membrane electrical potential and/or the trans-membrane pH gradient and thereby also the proton motive force, and eventually leads to cell death.

Bacteriocin-induced membrane-leakage and cell death may be prevented by immunity proteins that protect the bac-teriocin-producing bacteria from being killed by their own bacteriocins. It is not clear how immunity proteins protect cells from two-peptide bacteriocins, but they might act via association with the cell membrane, since structure predic-tions suggest that many immunity proteins have trans-membrane helices. The putative immunity proteins of the two-peptide bacteriocins plantaricin S [4, 117], brochocin-C [4, 121] and thermophilin 13 [4, 122] appear to contain two trans-membrane helices, whereas the putative immunity pro-teins of plantaricin E/F [4, 115], plantaricin J/K [4, 115], lactococcin MN [4, 123], and lactococcin G [4, 138] may

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 25

contain 4-5 trans-membrane helices. The number of trans-membrane helices thus seems to differ, but a common mechanism for bacteriocin-immunity involving interactions with membrane proteins (as is the case for the immunity pro-teins for the pediocin-like bacteriocins) may nevertheless exist.

The Two Peptides of Two-Peptide Bacteriocins Function as One Antimicrobial Entity

The individual peptides of two-peptide bacteriocins share characteristics with one-peptide bacteriocins in that they are usually (i) cationic, (ii) 30-50 residues long, (iii) hydropho-bic and/or amphiphilic, and are all (iv) synthesized with a 15-30 residue N-terminal leader sequence of the so-called double-glycine type that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter2 upon export of the peptides from cells [2, 4, 11, 12]. Moreo-ver, for some two-peptide bacteriocins, such as lactacin F [119], plantaricin E/F and plantaricin J/K [114], one or both peptides may individually display some – although low – antimicrobial activity. For many two-peptide bacteriocins, however, the individual peptides display no activity. For instance, the two peptides that constitute lactococcin G show no activity when tested individually at concentrations as high as 50 μM, but are active at 50 pM when combined [109, 133]. It should also be noted that a peptide from a two-peptide bacteriocin displays potent antimicrobial activity only when combined with the complementary peptide from the same two-peptide bacteriocin, or in some cases when combined with a peptide from a homologous two-peptide bacteriocin. For instance, the two peptides that constitute lactococcin G show no activity in combination with either the E- or F-peptide of plantaricin E/F or the J- or K-peptide of plantaricin J/K [114]. High activity is, however, obtained upon combining one of the lactococcin G peptides with the complementary peptide from lactococcin Q or enterocin 1071 [11, 113, 139], as these three two-peptide bacteriocins show extensive sequence similarities (about 57% sequence identity between lactococcin G and enterocin 1071, 88% identity between lactococcin G and Q, and 59% identity be-tween lactococcin Q and enterocin 1071 [11, 111, 113, 139]; Fig. (3A)).

The fact that both complementary peptides are required to obtain a potent antimicrobial effect and that the peptides only function together if they belong to the same two-peptide bacteriocin (or homologous bacteriocins) clearly indicates that the two peptides of two-peptide bacteriocins indeed function together as one antimicrobial entity, and should not simply be thought of as two synergistically-acting one-peptide bacteriocins. This is also consistent with results showing that (i) the genes encoding the two peptides of all two-peptide bacteriocins are next to each other on the same operon and the two peptides are thus produced in approxi-mately equal amounts, and that (ii) there is only one immu-

2 A novel feature of this ABC-transporter (its gene is either in the operon that contains

the bacteriocin and immunity genes or in a nearby operon) is an N-terminal extension

of about 150 residues that cleaves off the leader sequence at the C-terminal side of the

double-glycine motif [103]. As is the case for the pediocin-like bacteriocins, the leader

facilitates interaction with the ABC-transporter and might possibly also function to

keep the bacteriocin inactive until it has been secreted.

nity gene (in the same operon as the two bacteriocin genes) for each two-peptide bacteriocin [110-113, 115, 117-127, 138]. Moreover, circular dichroism (CD) structural studies of the three two-peptide bacteriocins lactococcin G, plantaricin E/F and plantaricin J/K [140, 141] indicate that their com-plementary peptides interact in a structure-inducing manner upon arrival at the target membrane. This is presumably also true for enterocin 1071 and lactococcin Q, since these two bacteriocins have more than 55% sequence identity with lactococcin G and thus similar three-dimensional structure and mode of action. The synergistic action of complementary peptides of two-peptide bacteriocins is thus apparently due to inter-peptide interactions, rather than to the two complemen-tary peptides acting separately at different sites on target-cells. It is not clear exactly at what stage this inter-peptide interaction occurs when peptides are exposed to sensitive cells. It presumably occurs, however, when the peptides come in contact with target cells, but before they become fully embedded in the membrane lipids. The CD studies showed that the structure-inducing inter-peptide interaction occurred when complementary peptides are added simulta-neously to membrane-like entities such as liposomes, but not when one peptide is added before the other nor when two liposome samples, each containing a peptide, are mixed [140]. It consequently seems that the peptides might first bind individually to an entity on the cell wall or membrane and then interact before penetrating further into the hydro-phobic part of the cell membrane. Such a sequence of events is consistent with results showing that toxicity is observed when sensitive cells are first treated with one lactococcin G peptide, washed and then treated with the other lactococcin G peptide, whereas no toxicity is detected when cells treated with one peptide are mixed with cells treated with the other peptide [134]. This suggests that both peptides can bind separately to the target-cell surface and remain in a "dormant state" without losing their potential bacteriocin activity, but they are unable to diffuse to another cell once bound to the cell surface [134].

Although two-peptide bacteriocins are clearly not simply two one-peptide bacteriocins that function in a synergistic manner, two-peptide bacteriocins might have evolved from two such one-peptide bacteriocins [11]. If two synergisti-cally-acting one-peptide bacteriocins were produced by the same bacteria, there might be a selection pressure for the enhancement of the synergistic effect - possibly at the ex-pense of the activity that the peptides have individually. This might in turn create selection pressure for genetically linking the two peptides, with the formation of a two-peptide bacte-riocin.

The Structure of Two-Peptide Bacteriocins

The three-dimensional structure of two-peptide (class-IIb) bacteriocins is not as well characterized as that of the pediocin-like (class-IIa) bacteriocins. Moreover, the two-peptide bacteriocins are much more diverse, judging from their primary structures, and are consequently expected to also be much more diverse with respect to their three-dimensional structure. CD and NMR structural studies have been carried out on the three two-peptide bacteriocins, lacto-coccin G, plantaricin E/F and plantaricin J/K [140-143]. CD studies show that the peptides of these three bacteriocins are

26 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

unstructured, with no structural interaction between com-plementary peptides, when in aqueous solution. Helical structuring occurs, however, when the peptides are individu-ally exposed to a more hydrophobic or membrane-like envi-ronment, such as when the peptides are in solutions contain-ing trifluoroethanol (TFE), micelles or liposomes [140, 141]. Moreover, additional structuring is obtained when comple-mentary peptides are mixed and then exposed to membrane-like entities such as liposomes, indicating that the peptides interact in a structure-inducing manner upon contact with target membranes [140, 141]. It seems that the target mem-brane must have a reasonable flat surface (i.e. liposomes or bilayers) for the inter-peptide interaction to take place, since the additional structuring is not observed in the presence of micelles.

Structure and Membrane-Orientation of Lactococcin G

Lactococcin G consists of the 39 residue peptide and the 35 residue peptide [109] (Fig. (3A)). Recent NMR-structural studies of these two peptides revealed that -helices are formed in both the N- (residues 3-21) and C-terminal (residues 24-34) halves of the peptide upon expo-sure to DPC micelles or TFE [142]. Helices are also formed in both the N- (residues 11-19) and C-terminal (residues 23-32) halves of the peptide in TFE, but only one helix, in the N-terminal half, was observed in DPC [142].

GxxxG-motifs are often involved in helix-helix interac-tions in membrane proteins [144]. The lactococcin G pep-tide has two such motifs (at residues 7 to 11 and residues 18 to 22; Fig. (3A)) and the peptide has one (at residues 18 to 22; Fig. (3A)). The GxxxG-motif in the peptide and the first one (residues 7-11) in the peptide are conserved in enterocin 1071 and lactococcin Q, and are thus likely to be of structural importance (Fig. (3A)). The high helical content combined with the presence of GxxxG-motifs suggests that the and peptides of lactococcin G, and the homologous two-peptide bacteriocins lactococcin Q and enterocin 1071, may engage in helix-helix interactions. It has been proposed that the and peptides of these three bacteriocins interact through helix-helix interactions involving the GxxxG-motif at residues 7 to 11 in their peptide and the GxxxG-motif at residues 18 to 22 in their peptide. The proposed structural model entails that the and peptides in the helix-helix structure lie in a parallel orientation and in a staggered fash-ion relative to each other [142] (Fig. (4)). This interaction would generate and/or stabilize helical structuring in the somewhat flexible GxxxG-motifs and possibly also in the more unstructured or flexible segments between the N- and C-terminal helical regions in the and peptides. This may in turn explain the increased helical content observed by CD spectroscopy when complementary peptides are simultane-ously exposed to target membranes [140-142].

The proposed structural model (Fig. (4)) may explain results that have been obtained upon analyzing lactococcin G and enterocin 1071 by site-directed mutagenesis [139]. As mentioned above, these two bacteriocins have similar pri-mary structures (about 57% sequence identity), but one con-spicuous difference between them is that enterocin 1071 has a negative charge (which is absent in lactococcin G and Q) at the N-terminus of the peptide, whereas lactococcin G (and Q) has a negative charge (which is absent in enterocin 1071)

at position 10 in the -peptide (Fig. (3A)). Analysis of these two bacteriocins by site-directed mutagenesis revealed that the detrimental effect of removing the N-terminal negative charge in the enterocin 1071 peptide is neutralized by in-troducing a negative charge at position 10 in the enterocin peptide. These two residue-positions (i.e. residue-1 and -10 in the and peptide, respectively) are near each other in the proposed structure (Fig. (4)), and this could explain why a negative charge in residue-1 in the peptide (as in entero-cin 1071) is functionally equivalent to a negative charge in residue-10 in the peptide (as in lactococcin G and Q).

The proposed structural model (Fig. (4)) may also ex-plain site-directed mutagenesis results that indicate that the lactococcin G immunity protein recognizes both a sequence in the N-terminal region (residues 1-16) of the peptide and a sequence on the C-terminal side of residue 10 in the pep-tide (Oppegård et al., in preparation). In the proposed struc-tural model, the regions in the (residues 1-16) and (resi-dues 12-27) peptides are adjacent to each other, and may consequently interact simultaneously with the immunity pro-tein. The amino acid residues in the GxxxG-motifs in lacto-coccin G have also been altered by site-directed mutagenesis. Consistent with being involved in helix-helix interactions, the results revealed that it was highly detrimental to replace the glycine residues in the GxxxG-motifs in the and pep-tides with a larger residue that interfere with close helix-helix contact (Oppegård, C.; Schmidt, J.; Kristiansen, P.E. and Nissen-Meyer, J. (2008) Biochemistry, 47(18), 5242-5249).

The clustering of positively charged residues at the C-terminal end of the peptide of enterocin 1071 and lacto-coccin G and Q is striking (Fig. (3A)), and it has been pro-posed that this region is forced through the target-cell mem-brane by the trans-membrane potential (negative inside), thereby orienting the positively charged C-terminal region of the peptide (residues 32-39) inside the cell and the trypto-phan-rich N-terminal region of the peptide (residues 1-10) in the outer interface part of the cell membrane (Fig. (4)). Positioning the tryptophan-rich N-terminal region of the peptide in the interface part of the membrane is consistent with mutagenesis analysis showing that any one of the three tryptophan residues (Trp-3, Trp-5 and Trp-8) in the N-terminal part of the lactococcin G peptide can be replaced with either an aromatic (Tyr, Phe), hydrophilic (Arg) or hy-drophobic (Leu) residue without a dramatic reduction in ac-tivity (Oppegård, C.; Schmidt, J.; Kristiansen, P.E. and Nis-sen-Meyer, J. (2008) Biochemistry, 47(18), 5242-5249), sug-gesting that this part of the peptide is neither embedded in the highly hydrophobic core of the membrane nor com-pletely in the hydrophilic outer environment. The proposed orientation of the and peptides in membranes (i.e. that N-termini of the peptides face the cells outside) is also con-sistent with results showing that fusion proteins consisting of the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminal residues of either the lactococcin G or peptide exhibit bacteriocin activity in combination with the complementary peptide. These re-sults suggest that the N-termini of the peptides do not pene-trate through the target cell membrane, and thus remain ex-ternally. This might, however, be an over interpretation of the results, since the fused -peptide was about 5,000-fold

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 27

Fig. (3). Amino acid sequences of two-peptide bacteriocins. (A) Sequence alignment of the and peptides of lactococcin G (LcnG),

lactococcin Q (LcnQ) and enterocin 1071 (Ent), where residues that are the same in at least two of the peptides are colored in red, residues

that are similar (i.e. conservative alteration) are in blue, and residues that are quite different from each other are in black. The GxxxG-motifs

are shaded in grey. (B) Sequences| of other two-peptide bacteriocins, where GxxxG-motifs are shaded in black. The peptides of plantaricin

S and NC8 do not have GxxxG-motifs, but contain similar AxxxA- and SxxxS-motifs (not shaded). References for sequences are as follows:

lactococcin G [109], lactococcin Q [113], enterocin [110], plantaricin E/F and J/K [114, 115], plantaricin S [117], plantaricin NC8 [118],

lactacin F [119], brochocin C [121], thermophilin 13 [122], ABP-118 [124], salivaricin P (differs from ABP-118 in only two residues) [125],

mutacin IV [126], and lactocin 705 [127]. See reference [123] for sequence of lactococcin MN. Leucocin H [130] and lactococcin MMT24

[132] are not included in the figure as they have not been completely sequenced.

28 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

Fig. (4). A cartoon depiction of the proposed structural model of lactococcin G and its orientation in membranes. The cylinders indi-

cate helical regions and the indicated trans-membrane helical regions in the and peptides are thought to form a coil-coil structure where

G7 and G11 in the GxxxG-motif in the peptide are in contact with, respectively, G18 and G22 in the GxxxG-motif in the peptide. The

peptides lie in a parallel orientation with their C-terminal ends on the cytoplasmic side of the membrane. Mutagenesis studies suggest that the

three tryptophan residues (W3, W5 and W8) in the N-terminal part of the peptide position themselves in the (outer) interface region, while

the five basic C-terminal residues (R35, K36, K37, K38, and H39) in the peptide are thought to position themselves on the cytoplasmic side

of the membrane. In the model, the negatively charged residue (D10) in the peptide is near the N-terminal residue in the peptide. Mutage-

nesis results reveal that the negative charge at position 10 (D10) (this negative charge is absent in enterocin peptide) is functionally equiva-

lent to the negative charge at position 1 (E1) in the enterocin peptide.

less potent than the un-fused -peptide and the fused -peptide was 20,000- to 30,000-fold less active than the un-fused -peptide (Rogne and Oppegård, unpublished results).

Structure of Plantaricin E/F

The three dimensional structures of the E (33 residues) and F (34 residues) peptides that constitute plantaracin E/F have also recently been analysed by NMR spectroscopy [143]. In the presence of DPC micelles, the E peptide forms two -helix-like regions (residues 10 to 21 and 25 to 31) separated by a flexible GxxxG-motif (residues 20 to 24), whereas the F peptide forms one long helix from residue 7 to 32, with a kink and slightly more flexible region around Pro-20 [143]. The E peptide has altogether two putative helix-helix interaction GxxxG-motifs, one at residue 5 to 9 and one at residue 20 to 24 (between the helices), while the F peptide has one such motif, at residue 30 to 34. It has been proposed for plantaricin E/F that its two peptides also interact in a par-allel and staggered fashion relative to each other and form a helix-helix structure involving the GxxxG-motifs [143].

Putative Helix-Helix Interaction Motifs are Common in Two-Peptide Bacteriocins

It should be noted that nearly all presently characterized peptides that are part of two-peptide (class-IIb) bacteriocins contain GxxxG-motifs (Fig. (3A and B)), suggesting that membrane-penetrating helix-helix structures formed by two peptides might possibly be a common structure in most, if not all, two-peptide bacteriocins. Such a structure might in-teract with an integrated membrane protein, somewhat

analogous to what seems to be the case for the pediocin-like (class-IIa) bacteriocins. As discussed above, the pediocin-like bacteriocins bind - apparently via their helical region that penetrates into membranes - to a part of the mannose phosphotransferase system that is embedded in the cell membrane. Interestingly, also the non-pediocin-like class-IId bacteriocin, lactococcin A, has been shown to interact to a part of the mannose phosphotransferase system that is em-bedded in the cell membrane [87] (see section below on class-IId bacteriocins). Helical interactions between peptide bacteriocins and integrated membrane (transport) proteins might thus be a common mechanism whereby peptide bacte-riocins cause membrane-leakage. There may, however, be some exceptions. CD and NMR studies conducted on bro-chocin-C suggest a high content of -sheet structure [145], and this may possibly also be the case for thermophilin 13, since it has marked sequence similarity with brochocin-C [121, 122]. However, also these two bacteriocins contain several GxxxG-motifs that suggest helix-helix interactions between their complementary peptides. The structures of brochocin-C and thermophilin 13 must consequently be ana-lysed in more detail before it can be concluded whether or not they in fact have a high content of -sheet structure.

Use of Site-Directed Mutagenesis to Identify Regions In-volved in Target-Cell Specificity and Interactions with

Immunity Proteins

Although there is great sequence similarity between lac-tococcin G [109, 133, 134, 140,], lactococcin Q, [113] and

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 29

enterocin 1071 [110-112] (Fig. (3A)), they nevertheless dif-fer in their relative potencies to different target-cells [11, 113, 139]. The three bacteriocins consequently represent a good system for correlating the structure of two-peptide bac-teriocins with target-cell specificity, potency, and interac-tions with immunity proteins. Bacteriocins that are hybrids of lactococcin G and enterocin 1071 have been constructed by mutating the lactococcin G gene in a step-wise fashion such that it finally becomes identical to the enterocin 1071 gene [139]. More than 15 variants of each of the lactococcin G peptides were thereby constructed. By measuring the po-tency against various target-cells of different combinations of these peptide constructs, the specificity determining re-gions in lactococcin G and enterocin 1071 was identified [139]. Taken together, the results suggest that the peptide of these bacteriocins is especially important in determining the target-cell specificity.

The lactococcin G immunity protein protects lactococci against lactococcin G, but not against enterocin 1071 (Op-pegård et al., in preparation). However, the lactococcin G immunity protein does protect enterococci against enterocin 1071 (Oppegård et al., in preparation). The immunity protein thus differentiates between similar bacteriocins, but its abil-ity to do so is target-cell dependent. This clearly indicates that its functionality depends on a cellular component, as is the case with the pediocin-like immunity proteins that inter-act rather specifically with their cognate bacteriocins indi-rectly via the mannose phosphotransferase permease [87] (see above section dealing with the pediocin-like bacteriocins). The efficiency with which the lactococcin G immunity pro-tein protected lactococci strains against the various lactococ-cin G/enterocin 1071 hybrids has been analyzed in order to identify regions in the and peptides that are specifically recognized by the immunity protein. The results revealed that sequences in the N-terminal region (residues 1-16) of the

peptide and on the C-terminal side of residue 10 in the -peptide were specifically (but possibly indirectly via a cell component) recognized by the immunity protein. As dis-cussed above in the section on the structure of lactococcin G, these results are consistent with the proposed structural model for lactococcin G, since the N-terminal part of the -peptide (residues 1-16) overlaps and forms a helix-helix structure with a region (residues 12-27) which is on the C-terminal side of residue 10 in the peptide (Fig. (4)).

THE CYCLIC (CLASS-IIC) BACTERIOCINS

The cyclic bacteriocins (recently reviewed in [146]) whose N- and C-termini are covalently linked are placed in class-IIc according to Cotter et al. [6], in class-III according to Franz et al. [147], and in class-V according to Kemper-man et al. [148]. At least 7 cyclic bacteriocins produced by Gram-positive bacteria have been characterized [148-163] (Table 1). They are all cationic (except for subtilosin A) and relatively hydrophobic, and they range in size from 3400 to 7200 Da. All cyclic bacteriocins whose mode of action has been characterized render the target-cell membrane perme-able to small molecules and thereby disrupt the proton mo-tive force, which eventually results in cell death. The advan-tage of the cyclic structure of these bacteriocins is not en-tirely clear, but it probably functions to stabilize the three-dimensional structure that is required for the bacteriocins’

antibacterial activity. Interestingly, a heterologously Es-cherichia coli-expressed non-cyclic version of gassericin A has 170-fold lower activity than the natural circular form of the bacteriocin [154]. The cyclic structure presumably also renders the bacteriocins more resistant to proteolysis.

Enterocin AS-48 (also termed enterocin-4, -EFS2, and bacteriocin-21) was the first cyclic bacteriocin to be identi-fied and it is the one that is best characterized [149-151, 167-169]. It contains 70 residues and is produced by several strains of Enterococcus faecalis and E. faecium [149, 164-166, 170, 171]. It is membrane permeabilizing and has a broad-range antibacterial spectrum, being active against many Gram-positive and some Gram-negative bacteria [151]. NMR structural analysis of the peptide in aqueous solution at pH 3 revealed a globular arrangement of five -helices that enclose a compact hydrophobic core [168]. Un-der these conditions, the bacteriocin is monomeric, but it forms dimers when the pH is between 4.5 and 8.5 [151, 169]. The crystal structure of the dimeric form of enterocin AS-48 at pH 4.5 and 7.5 revealed that the structure of the protomers constituting the dimeric form is overall the same as the NMR structure of the monomeric form [151, 169]. The protomers, however, interact with each other in two somewhat different ways, and two different crystal structures of the dimeric form are consequently observed; a water-soluble and a membrane-bound form. Two hydrophobic helices, which are partially hidden in the water-soluble form, apparently become ex-posed when the water-soluble form switches to the mem-brane-bound form at the membrane surface, and this may then permit insertion of the bacteriocin dimer into the target-membrane [151, 169].

Gassericin A produced by Lactobacillus gasseri LA39 [152-154] and reutericin 6 produced by Lactobacillus reuteri LA6 [155, 156] contain 58 residues and have identical amino acid sequences. The former seems, however, to contain two D-alanine residues, whereas the latter apparently contains only one [156]. This might explain why they differ in activ-ity (gassericin A seems to have a somewhat broader antibac-terial spectrum than reutericin 6) and why their CD spectra did not completely coincide [156]. Their three-dimensional structures are thus not necessarily completely identical, al-though the CD spectra revealed that both peptides are mainly composed of alpha-helices. It can not be excluded, however, that the apparent difference in activity and CD spectra may be due to other structural differences. The two peptides could for instance differ with respect to the configuration (trans or cis) of the peptide bond on the N-terminal side of a proline residue present in both bacteriocins, as the configuration of such a peptide bond influences the structure and activity of antimicrobial peptides [172].

Three of the presently identified cyclic bacteriocins are non-LAB bacteriocins, but two of these, circularin A and butyrivibriocin AR10, are homologous to cyclic LAB bacte-riocins. Circularin A is produced by a Clostridium beijer-inckii strain, contains 69 residues, and shows 60% sequence similarity and 30% identity to enterocin AS-48 [148, 154]. Butyrivibriocin AR10 is produced by a Butyrivibrio fibrisol-vens strain, contains 58 residues, and shows sequence simi-larity to gassericin A, reutericin 6 and acidocin B [154, 157, 158]. In contrast, subtilosin A, produced by a Bacillus subtil-

30 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

lis strain [159], is very different from all the other cyclic bacteriocins. It contains only 35 residues and is extensively post-translationally modified. It has three cross links formed between the sulphurs of three cysteine residues (Cys-13, Cys-7 and Cys-4) and the -carbon atoms of, respectively, two phenylalanine residues and one threonine residue (Phe-22 , Thr-28 and Phe-31, respectively) [159, 160]. NMR structural analysis revealed a twisted bowl-like structure, with most amino acid residue side chains pointing toward the solvent.

THE ONE-PEPTIDE NON-PEDIOCIN-LIKE LINEAR

(CLASS-IID) BACTERIOCINS

The linear non-pediocin-like one-peptide bacteriocins that show no sequence similarity to the pediocin-like bacte-riocins are placed in class-IId according to the classification proposed by Cotter et al. [6]. These bacteriocins were earlier placed in class-IIc or included in class-IIa along with the pediocin-like bacteriocins. Table 2 presents a list of class-IId bacteriocins [173-205]. The ones that are best characterized will be described below.

Lactococcin A

Lactococcin A, produced by some Lactococcus lactis strains, was among the first of the class-IId bacteriocins to be isolated and is the one that is best characterized [173, 174, 209]. It is initially synthesized as a 75-residue pre-bacteriocin that consists of a 21-residue double-glycine type leader sequence and the cationic 54-residue mature bacterio-cin [173]. The mature bacteriocin has a relatively narrow activity spectrum. It increases the permeability of target-cell membranes in a voltage-independent and protein-mediated manner, and thereby dissipates the proton motive force

[209]. As is the case for the pediocin-like (class-IIa) bacterio-cins, lactococcin A binds to the mannose phosphotransferase permease, apparently the part of the permease that is embed-ded in the target-cell membrane [87]. The lactococcin A immunity protein, which is partly associated with the plasma membrane within cells [210, 211], recognizes and binds strongly to the lactococcin A-permease complex, thereby presumably preventing lactococcin A-induced cell-killing [87]. Interactions between lactococcin A and the mannose phosphotransferase permease thus apparently alter the conformation of the permease in a manner that results in membrane-leakage, and this leakage is blocked by the bind-ing of an immunity protein to the bacteriocin-permease com-plex.

Enterococcal Bacteriocins

Several enterococcal class-IId bacteriocins have been iden-tified and characterized. Enterocin EJ97, produced by En-terococcus faecalis EJ97, is a cationic and hydrophobic 44-residue bacteriocin that is synthesized without an N-terminal leader sequence [175]. It is active against several Gram-positive bacteria, including enterococci and species of Bacil-lus, Listeria, and Staphylococcus aureus [212]. Enterocin B, produced by Enterococcus faecium T136, contains 53 resi-dues after removal of the 18-residue double-glycine type leader sequence. The bacteriocin has a rather wide activity spectrum, being active against listeria, staphylococci, and most LAB that have been tested [176]. It has 47% sequence identity to carnobacteriocin A (identical to piscicolin 61 [177]), which is a 53-residue bacteriocin produced by Carnobacterium piscicola LV17A [178]. Bac32, produced by Enterococcus faecium strains, is a 70-residue bacteriocin with a rather narrow activity spectrum [179]. Enterocin L50A, L50B and enterocin Q are all produced by

Table 1. Cyclic (Class-IIc) Bacteriocins

Bacteriocins Producing strain References

Enterocin AS-481 Enterococcus faecalis S-48 [149-151]

Gassericin A Lactobacillus gasseri LA39 [152-154]

Reutericin 62 Lactobacillus reuteri LA6 [155-156]

Circularin A Clostridium beijerinckii ATCC 25752 [148, 154]

Butyrivibriocin AR10 Butyrivibrio fibrisolvens AR10 [157-158]

Subtilosin A Strains of Bacillus subtillis [159-160]

Acidocin B3 Lactobacillus acidophilus M46 [161]

Uberolysin4 Streptococcus uberis strain 42 [162]

Acidocin D200795 L. acidophilus DSM 20079 [163]

1 Enterocin AS-48 is identical to enterocin 4, produced by E. faecalis INIA 4 [164], enterocin EFS2 produced by E. faecalis EFS2 [165], and bacteriocin-21 produced by an E. fae-

calis strain [166]. 2 Reutericin 6 has identical amino acid sequence as gassericin A, but it has been reported to contain one D-alanine, whereas gassericin A has been reported to contain two [156]. 3 The circular structure of acidocin B has not yet been definitely established, but it has 98% sequence identity to gassericin A and reutericin 6. 4 Uberolysin contains 70 residues and has been shown to induce lysis of target bacteria. 5 Acidocin D20079 is thought to be cyclic as it is resistant to Edman degradation. Moreover, C-terminal sequencing was unsuccessful, and treatment with carboxypeptidases A and B

did not cleave off any residues. Proteolytic cleavage followed by sequencing of generated fragment yielded a partial sequence consisting of 39 of a totally estimated 65 residues

[163].

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 31

Table 2. The One-Peptide Non-Pediocin-Like Linear (Class-IId) Bacteriocins

Bacteriocins Producing strains References

Lactococcin A Strains of Lactococcus lactis [173, 174]

Enterocin EJ97 Enterococcus faecalis EJ97 [175]

Enterocin B Enterococcus faecium T136 [176]

Carnobacteriocin A1 Carnobacterium piscicola LV17A [178]

Bac32 E. faecium VRE200 [179]

Enterocin L50A2 E. faecium L50 [131]

Enterocin L50B2 E. faecium L50 [131]

Enterocin Q E. faecium L50 [180]

Enterocin RJ-113 E. faecalis RJ-11 [183]

Aureocin A704 Staphylococcus aureus A70 [184]

Aureocin A535 S. aureus A53 [185]

Lacticin Q5 L. lactis QU5 [186]

Lacticin Z5 L. lactis QU14 [187]

BHT-B5 Strains of Streptococcus rattus and mutans [188]

Lactococcin B L. lactis subsp. cremoris 9B4 [189]

Acidocin A6 Lactobacillus acidophilus TK9201 [190]

Bacteriocin OR-76 Lactobacillus salivarius NRRL B-30514 [191]

Divergicin 7507 Carnobacterium divergens 750 [192]

Weissellicin 1108 Weisella cibaria 110 [193]

Acidocin 1B9 L. acidiphilus GP1B [194]

Acidocin CH510 L. acidophilus CH5[ [195]

Cripacin A11 Lactobacillus crispatus JCM 2009 [196]

Lacticin RM12 L. lactis subsp. lactis EZ26 [197]

Micrococcin GO513 Micrococcus sp. GO5 [198]

LsbA and LsbB14 Natural isolate of L. lactis [199]

Mesenterocin 52B15 Leuconostoc mesenteroides subsp. Mesenteroides

FR52

[200]

Mesentericin B10515 L. mesenteroides Y105 [201]

Dextranicin 2415 L. mesenteroides subsp. dextranicum J24 [202]

Leucocin B-TA33a15 L. mesenteroides TA33a [203]

Lactococcin 97216 L. lactis IPLA 972 [204]

Bovicin 25517 Streptococcus gallolyticus LRC0255 [205]

1 Carnobacteriocin A is identical to piscicolin 61 produced by C. piscicola LV61 [177]. 2 Enterocin L50A and L50B show 72% sequence identity. The two peptides act synergistically, but are not considered to constitute a bona fide two-peptide bacteriocin since the two

peptides have similar sequences and have potent antimicrobial activity when assayed individually. These two peptides have also been isolated from E. faecium 6T1a [181], and nearly

identical peptides from E. faecalis MRR 10-3 [182]. 3 Enterocin RJ-11 has 75% sequence identity to enterocin L50A. 4 It is not clear if aureocin A70 is a novel four-peptide bacteriocin or simply four separate one-peptide bacteriocins with similar sequences (see text). 5 There is 47% sequence identity between lacticin Q and BHT-B, and 48% between lacticin Q and aureocin A53. Lacticin Q and Z differ in residues [187]. 6 Acidocin A and bacteriocin OR-7 have 70% sequence identity. They have some sequence similarity with the pediocin-like class-IIa bacteriocins, but are nevertheless placed among

the non-pediocin-like class-IId bacteriocins (se text).

32 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

(Legend Table 2) contd…..

7 Divergicin 750 is a cationic 34-residue peptide.

8 Only the 27 N-terminal residues of Weissellicin 110 have been sequenced. Its molecular mass is 3,488 Da. 9 Only 6 of the first 7 residues of acidocin have been sequenced. 10 The first 13 N-terminal amino acid residues of acidocin CH5 have been sequenced and found to be identical/similar to several bacteriocin-like peptides produced by other L. aci-

dophilus strains [206-208]. 11 Cripacin A is partially sequenced and has a molecular mass of 5393 Da. 12 The lacticin RM gene has been sequenced and encodes a putative 134-residue cationic peptide. 13 Only the first 7 N-terminal residues of micrococcin GO5 have been sequenced. 14 LsbA and LsbB are two cationic narrow spectrum bacteriocins. The former is a 44-residue rather hydrophobic peptide that is initially synthesized with an N-terminal leader se-

quence, while the latter is a 30-residue relatively hydrophilic peptide that is synthesized without a leader sequence. 15 Mesenterocin 52B and mesentericin B105, and possibly also dextranicin 24, are identical 32-residue bacteriocins that share 62% identity with the 31-residue bacteriocin leucocin B-

TA33a. 16 Lactococcin 972 is a cationic 66-residue hydrophilic bacteriocin, which may function by inhibiting cell wall synthesis rather than through membrane permeabilization. 17Bovicin 255 is a 56-residue peptide.

Enterococcus faecium L50 and synthesized without a leader sequence [131, 180]. Enterocin Q consists of 34 residues and has a rather narrow activity spectrum [180]. Enterocin L50A and L50B show 72% sequence identity to each other and contain 44 and 43 residues, respectively [131]. Although both of these bacteriocins have considerable antimicrobial activity when tested separately, synergism is observed against some target-cells when they are combined. These two bacteriocins have also been isolated from Enterococcus fae-cium 6T1a [181] and nearly identical peptides (one substitu-tion in L50A and two in L50B) have been isolated from En-terococcus faecalis MRR 10-3 [182]. The 44-residue bacte-riocin enterocin RJ-11 isolated from Enterococcus faecalis RJ-11 showed 75% sequence identity to enterocin L50A, but the antibacterial spectra seemed to be somewhat different [183].

Aureocin A70

Aureocin A70, produced by Staphylococcus aureus A70, is unusual in that it consists of four cationic, hydrophobic and unmodified 30- or 31-residue peptides that have exten-sive sequence similarities, but show no sequence similarities to other peptides [184]. The peptides are synthesized without a leader sequence, and the four genes that encode these pep-tides are in the same operon. Three of the four peptides dis-play antimicrobial activity alone. The fourth peptide could not be purified and its antimicrobial activity could thus not be evaluated [184], but its similarity to the other three pep-tides suggests that also this fourth peptide is active. It is not clear if these four peptides represent a four-peptide bacterio-cin or simply four separate (but related) one-peptide bacte-riocins, since it is not known if these peptides act synergisti-cally and or interact physically with each other. They might possibly be four independently-acting bacteriocins that differ somewhat in their antimicrobial spectrum, and by being pro-duced together they broaden the antimicrobial spectrum of Staphylococcus aureus A70.

Aureocin A53

Some Staphylococcus aureus strains also produce aureo-cin A53, a highly cationic and tryptophan-rich 51-residue bacteriocin, which has a formylated N-terminal methionine residue and is synhesized without a leader sequence [185]. It is relatively protease stable and seems to have a defined rigid structure in aqueous solution; significant parts are in helical (about 36%) and in -sheet (about 18%) conformations in

aqueous solution [185]. There is apparently no need of spe-cialized processing proteins for production of this bacterio-cin, as no genes encoding such proteins (for instance ABC-transporters, immunity proteins, modifying enzymes, and regulatory proteins) were found in the vicinity of the bacte-riocin gene [185]. The bacteriocin shows bactericidal activity against a broad range of LAB, Listeria monocytogenes, and many strains of Staphylococcus aureus. Its bactericidal activ-ity seems to be due to membrane disruption rather than from the formation of defined or target-mediated pores, since mi-cromolar concentrations of the bacteriocin are required to kill bacteria and induce efflux from cells and negatively charged liposomes [213]. This is in contrast to bacteriocins (such as the pediocin-like (class-IIa) and most two-peptide (class-IIb) bacteriocins) that function at nanomolar concen-trations and appear to act through the formation of pores upon binding with high affinity to specific receptors or dock-ing molecules. Aureocin A53 rapidly dissipated the mem-brane potential and induced rapid release of glutamate and Rb

+, and simultaneously stopped DNA, polysaccharide and

protein synthesis [213]. Aureocin A53 has 46-48% sequence identity to lacticin Q [186] and Z [187], two homologous 53-residue bacteriocins produced by Lactococcus lactis QU5 and QU14, respectively. Also lacticin Q and Z have formy-lated N-terminal methionine residues and are synthesized without leader sequences [186, 187]. Aureocin A53 has also some sequence similarity to BHT-B, a 44-residue bacteriocin that is also synthesized without a leader sequence and pro-duced by several Streptococcus rattus and S. mutans strains [188]. There is 47% sequence identity between BHT-B and lacticin Q.

Lactococcin B

Lactococcin B, produced by Lactococcus lactis subsp. cremoris 9B4, is a cationic, hydrophobic bacteriocin that dissipates the proton motive force and causes leakage of in-tracellular molecules [189]. It contains one cysteine residue (Cys-24), which must be in reduced state for the bacteriocin to be active [189]. Replacement of this residue with any other residue that does not have a positive charge resulted in bacteriocin variants that were more active, probably because the cysteine residue in the wild-type bacteriocin tends to become oxidized, which results in reduced activity [214]. It is somewhat surprising that the single cysteine residue is present in the wild type peptide, and the results suggest that the cysteine residue has an important function other than rendering the bacteriocin active.

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 33

Acidocin A and OR-7

Acidocin A, produced by Lactobacillus acidophilus TK9201 [190], and bacteriocin OR-7 [191], produced by Lactobacillus salivarius NRRL B-30514, contain 58 and 54 residues, respectively, and they are identical in about 70% of their residues. These two bacteriocins show sequence simi-larities with the pediocin-like bacteriocins in their N-terminal regions. Both acidocin A and bacteriocin OR-7 contain the N-terminal sequence K-T-Y-Y-G-T-N-G-V-H-C-T-K- which is nearly identical to the N-terminal sequences of the pedio-cin-like bacteriocins (Fig. (1)). However, the sequences in middle and C-terminal parts of acidocin A and bacteriocin OR-7 are overall different from the corresponding sequences found in the pediocin-like bacteriocins, and they are conse-quently not grouped with the pediocin-like class-IIa bacte-riocins.

Plantaricin 1.25

Plantaricin 1.25 , produced by Lactobacillus plantarum TMW1.25, is a cationic 53-residue bacteriocin [215]. The sequence of the first 28 amino acid residues is identical to the partial amino acid sequence of brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27 [216]. Plantaricin 1.25 has also some similarity to lactobin A [128] and amy-lovorin L471 [217]. Although presently classified and de-scribed as a one-peptide bacteriocin, plantaricin 1.25 (and thus possibly also brevicin 27, lactobin A and amylovorin L471) might be part of a two-peptide bacteriocin, since next to (and in the same operon) the gene encoding plantaricin 1.25 is a gene that encodes a 73-residue bacteriocin-like precursor consisting of an 18-residue double glycine leader peptide and a cationic 55-residue mature peptide termed plantaricin 1.25 [218]. Plantaricin 1.25 might consequently be the complementary peptide to plantaricin 1.25 . Interest-ingly, both peptides have several GxxxG-motifs that are commonly found in two-peptide bacteriocins. There is also a striking cluster of positively charged lysine residues in the N-terminal region of both peptides (the first 5 and 6 residues in, respectively, plantaricin 1.25 and plantaricin 1.25 are lysines), which suggests that the N-terminal end may be forced through the target-cell membrane by the trans-membrane potential (negative inside), thereby generating trans-membrane peptides that might then interact with cellu-lar integrated membrane proteins.

APPLICATIONS, FUTURE PERSPECTIVES AND CONCLUDING REMARKS

Nisin and pediocin PA-1 are presently used as bio-preservatives in food, the former being approved in over 40 countries for use as a food additive [6]. It is expected that we in coming years will see a number of other bacteriocins be-ing successfully used for preservation of food and animal feed and for treatment of infections. Moreover, it is likely that genetically modified bacteriocin variants with improved properties as judged from an applied point of view will be-come available. It should, however, be realized that rational design of new and improved bacteriocins is difficult, and requires detailed insight into the structure and mechanisms underlying the activity of bacteriocins. In fact, most altera-tions done on bacteriocins are detrimental or neutral. Amino acid substitutions reveal that residues important for activity

are generally distributed across the entire sequence of most bacteriocins [66, 73-75, 219, 220] and few residues are thus dispensable or may be altered without a detrimental effect. Some genetically modified bacteriocins that have properties that are as good as, or better, than the corresponding natural bacteriocins have nevertheless been constructed. Introducing a hairpin-stabilizing C-terminal disulfide bridge into the pediocin-like bacteriocin sakacin P broadened the target cell specificity and rendered the bacteriocin more thermostable [64]. Increasing the net positive charge of some bacteriocins has improved their potency somewhat, probably by causing greater binding to the negatively charged surface of target cells [73]. Replacement of the methionine residue with a hydrophobic residue has protected pediocin-like and two-peptide bacteriocins from inactivation by oxidation, without causing a marked reduction in bacteriocin activity [65, 142], and replacement of the cysteine residue in lactococcin B with any other residue that does not have a positive charge re-sulted in lactococcin B variants that were more active than the wild-type bacteriocin [214].

Structure-function analysis is not only invaluable for ra-tional design of bacteriocin variants with properties that make them especially useful for medical and biotechnologi-cal applications, but gives us detailed insight at a molecular level into features that are important for their mode of action and potency. As mentioned earlier, extensive structure-function and biochemical studies of the pediocin-like bacte-riocins reveal that they kill sensitive cells by interacting with the membrane-associated mannose phosphotransferase per-mease. A part, presumably a helical segment, of these bacte-riocins binds to permease-subunits that are embedded in the membrane, thereby imposing structural alterations in the permease - alterations that lead to membrane-leakage. The cognate immunity proteins sense these alterations and pre-vent membrane-leakage upon binding to the bacteriocin-permease complex [87]. The facts that a completely different bacteriocin, lactococcin A, has been shown to function in a similar manner [87] and that membrane-active helical struc-tures are found in several peptide bacteriocins (including two-peptide bacteriocins), suggest that many membrane-active peptide bacteriocins might induce membrane-leakage through basically the same mechanism. The common theme in such a mechanism is that membrane-leakage (directly or indirectly) results from conformational alterations in inte-grated membrane (transport) proteins, and that these confor-mational alterations are induced by the binding of a mem-brane-penetrating (probably helical) part of the bacteriocin to these proteins. If this is a common mechanism of action for peptide bacteriocins, more detailed insight into structure-function relationships of peptide bacteriocins may enable rational design of novel peptides that penetrate into mem-branes and bind to and interfere with the functioning of spe-cific membrane proteins.

ACKNOWLEDGMENTS

The authors have been supported by the Norwegian Re-search Council.

REFERENCES

[1] Boman, H.G. (1995) Annu. Rev. Immunol., 13, 61-92.

34 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

[2] Nissen-Meyer, J. and Nes, I.F. (1997) Arch. Microbiol., 167(2), 67-

77. [3] Zasloff, M. (2002) Nature, 415(6870), 389-395.

[4] Nes, I.F.; Holo, H.; Fimland, G.; Hauge, H.H. and Nissen-Meyer, J. (2002) in Peptide antibiotics: discovery, modes of action and ap-

plication, (Dutton, C.J.; Haxell, M.A.; McArthur, H.A.I. and Wax, R.G., Eds.). Marcel Dekker, New York, pp. 81-115.

[5] Pag, U. and Sahl, H.-G. (2002) Curr. Pharm. Des., 8(9), 815-833. [6] Cotter, P.D.; Hill, C. and Ross, R.P. (2005) Nature Rev. Microbiol.,

3(10), 777-788. [7] Corr, S.C.; Li, Y.; Riedel, C.U.; O’Toole, P.W.; Hill, C. and

Gahan, C.G.M. (2007) Proc. Natl. Acad. Sci. USA, 104(18), 7617-7621.

[8] Skaugen, M.; Nissen-Meyer, J.; Jung, G.; Stevanovic, S.; Sletten, K.; Mørtvedt Abildgaard, C.I. and Nes, I.F. (1994) J. Biol. Chem.,

269(44), 27183-27185. [9] Drider, D.; Fimland, G.; Héchard, Y.; McMullen, L.M. and

Prévost, H. (2006) Microbiol. Mol. Biol. Rev., 70(2), 564-582. [10] Fimland, G.; Johnsen, L.; Dalhus, B. and Nissen-Meyer, J. (2005).

J. Peptide Sci., 11(11), 688-696. [11] Oppegård, C.; Rogne, P.; Emanuelsen, L.; Kristiansen, P. E.; Fim-

land, G. and Nissen-Meyer. J. (2007) J. Mol. Microbiol. Biotech-nol., 13(4), 210-219.

[12] Garneau, S.; Martin, N.I. and Vederas, J.C. (2002) Biochimie, 84(5-6), 577-592.

[13] Eijsink, V.G.H.; Skeie, M.; Middelhoven, P.H.; Brurberg, M.B. and Nes, I.F. (1998) Appl. Environ. Microbiol., 64(9), 3275-3281.

[14] Ingham, A.; Ford, M.; Moore, R.J. and Tizard M. (2003) J. Antimi-crob. Chemother., 51(6), 1365-1371.

[15] Hastings, J.W.; Sailer, M.; Johnson, K.; Roy, K.L.; Vederas, J.C. and Stiles, M. (1991) J. Bacteriol., 173(23), 7491-7500.

[16] Tichaczek, P.S.; Nissen-Meyer, J.; Nes, I.F.; Vogel, R.F. and Hammes, W.P. (1992) Syst. Appl. Microbiol., 15(4), 460-468.

[17] Tichaczek, P.S.; Vogel, R.F. and Hammes, W.P. (1994) Microbiol-ogy, 140(2), 361-367.

[18] Hühne, K.; Axelsson, L.; Holck, A. and Kröckel, L. (1996) Micro-biology, 142(6), 1437-1448.

[19] Tichaczek, P.S.; Vogel, R.F. and Hammes, W.P. (1993) Arch. Microbiol., 160(4), 279-283.

[20] Holck, A.; Axelsson, L.; Birkeland, S.E.; Aukrust, T. and Blom, H. (1992) J. Gen. Microbiol., 138(12), 2715-2720.

[21] Axelsson, L. and Holck, A. (1995) J. Bacteriol., 177(8), 2125-2137.

[22] Héchard, Y.; Dérijard, B.; Letellier, F. and Cenatiempo, Y. (1992) J. Gen. Microbiol., 138(12), 2725-2731.

[23] Fremaux, C.; Héchard, Y. and Cenatiempo, Y. (1995) Microbiol-ogy, 141(7) 1637-1645.

[24] Fleury, Y.; Dayem, M.A.; Montagne, J.J.; Chaboisseau, E.; Le Caer, J.P.; Nicolas, P. and Delfour, A. (1996) J. Biol. Chem.,

271(24), 14421-14429. [25] Nieto Lozano, J.C.; Nissen-Meyer, J.; Sletten, K.; Peláz, C. and

Nes, I.F. (1992) J. Gen. Microbiol., 138(9), 1985-1990. [26] Marugg, J.D.; Gonzalez, C.F.; Kunka, B.S.; Ledeboer, A.M.; Pucci,

M.J.; Toonen, M.Y.; Walker, S.A.; Zoetmulder, L.C.M. and Van-denbergh, P.A. (1992) Appl. Environ. Microbiol., 58(8), 2360-

2367. [27] Henderson, J.T.; Chopko, A.L. and van Wassenaar, P.D. (1992)

Arch. Biochem. Biophys., 295(1), 5-12. [28] Motlagh, A.M.; Buhnia, A.K.; Szostek, F.; Hansen, T.R.; Johnson,

M.C. and Ray, B. (1992) Lett. Appl. Microbiol., 15(2), 45-48. [29] Aymerich, T.; Holo, H.; Håvarstein, L.S.; Hugas, M.; Garriga, M.

and Nes, I.F. (1996) Appl. Environ. Microbiol., 62(5), 1676-1682. [30] Métivier, A.; Pilet, M.F.; Dousset, X.; Sorokine, O.; Anglade, P.;

Zagorec, M.; Piard, J.C.; Marion, D.; Cenatiempo, Y. and Fremaux, C. (1998) Microbiology, 144(10), 2837-2844.

[31] Tahiri, I.; Desbiens, M.; Benech, R.; Kheadr, E.; Lacroix, C.; Thi-bault, S.; Ouellet, D. and Fliss, I. (2004) Int. J. Food Microbiol.,

97(2), 123-136. [32] Le Marrec, C.; Hyronimus, B.; Bressollier, P.; Verneuil, B. and

Urdaci, M.C. (2000) Appl. Environ. Microbiol., 66(12), 5213-5220. [33] Yamazaki, K.; Suzuki, M.; Kawai, Y.; Inoue, N. and Montville, T.

(2005) Appl. Environ. Microbiol., 71(1), 554-557. [34] Kalmokoff, M.L.; Banerjee, S.K.; Cyr, T.; Hefford, M.A. and Glee-

son, T. (2001) Appl. Environ. Microbiol., 67(9), 4041-4047.

[35] Kawamoto, S.; Shima, J.; Sato, R.; Eguchi, T.; Ohmomo, S.; Shi-

bato, J.; Horikoshi, N.; Takeshita, K. and Shameshima, T. (2002) Appl. Environ. Microbiol., 68(8), 3830-3840.

[36] Bennik, M.H.J.; Vanloo, B.; Brasseur, R.; Gorris, L.G.M. and Smid, E.J. (1998) Biochim. Biophys. Acta., 1373(1), 47-58.

[37] Jack, R.W.; Wan, J.; Gordon, J.; Harmark, K.; Davidson, B.E.; Hillier, A.J.; Wettenhall, R.E.H.; Hickey, M.W. and Coventry, M.J.

(1996) Appl. Environ. Microbiol., 62(8), 2897-2903. [38] Bhugaloo-Vial, P.; Dousset, X.; Metivier, A.; Sorokine, O.; Ang-

lade, P.; Boyaval, P. and Marion, D. (1996) Appl. Environ. Micro-biol., 62(12), 4410-4416.

[39] Vaughan, A.; Eijsink, V.G.H.; O’Sullivan, T.F.; O’Hanlon, K. and van Sinderen, D. (2001) J. Appl. Microbiol., 91(1), 131-138.

[40] Fimland, G.; Sletten, K. and Nissen-Meyer, J. (2002) Biochem. Biophys. Res. Commun., 295(4), 826-827.

[41] Ferchichi, M.; Frère, J.; Mabrouk, K. and Manai, M. (2001) FEMS Microbiol. Lett., 205(1), 49-55.

[42] Simon, L.; Fremaux, C.; Cenatiempo, Y. and Berjeaud, J.M. (2002) Appl. Environ. Microbiol., 68(12), 6416-6420.

[43] Atrih, A.; Rekhif, N.; Moir, A.J.G.; Lebrihi, A. and Lefebvre, G. (2001) Int. J. Food Microbiol., 68(1-2), 93-104.

[44] van Reenen, C.A.; Chikindas, M.L.; van Zyl, W.H. and Dicks, L.M.T. (2003) Int. J. Food Microbiol., 81(1), 29-40.

[45] Quadri, L.E.N.; Sailer, M.; Roy, K.L.; Vederas, J.C. and Stiles, M.E. (1994) J. Biol. Chem., 269(16), 12204-12211.

[46] Cintas, L.M.; Casaus, P.; Håvarstein, L.S.; Hernández, P.E. and Nes, I.F. (1997) Appl. Environ. Microbiol., 63(11), 4321-4330.

[47] Tomita, H.; Fujimoto, S.; Tanimoto, K. and Ike, Y. (1996) J. Bac-teriol., 178(12), 3585-3593.

[48] Del Campo, R.; Tenorio, C.; Jiménez-Diaz, R.; Rubio, C.; Gomez-Lus, R.; Baquero, F. and Torres C. (2001) Antimicrob. Agents

Chemother., 45(3), 905-912. [49] De Kwaadsteniet, M.; Fraser, T.; Van Reenen, C.A. and Dicks,

L.M.T. (2006) Appl. Environ. Microbiol., 72(7), 4761-4766. [50] Diep, D.B.; Godager, L.; Brede, D. and Nes, I.F. (2006) Microbiol-

ogy, 152(6), 1649-1659. [51] Eguchi, T.; Kaminaka, K.; Shima, J.; Kawamoto, S.; Mori, K.;

Choi, S.-H.; Doi, K.; Ohmomo, S. and Ogata, S. (2001) Biosci. Biotechnol. Biochem., 65(2), 247-253.

[52] Xiraphi, N.; Georgalaki, M.; Van Driessche, G.; Devreese, B.; Van Beeumen, J.; Tsakalidou, E.; Metaxopoulos, J. and Drosinos, E.H.

(2005) Antonie Van Leeuwenhoek, 89(1), 19-26. [53] Kang, J.H. and Lee, M.S. (2005) J. Appl. Microbiol., 98(5), 1169-

1176. [54] Fimland, G.; Blingsmo, O.R.; Sletten, K.; Jung, G.; Nes, I.F. and

Nissen-Meyer, J. (1996) Appl. Environ. Microbiol., 62(9), 3313-3381.

[55] Chikindas, M.L.; García-Garcerá, M.J.; Driessen, A.J.M.; Lede-boer, A.M.; Nissen-Meyer, J.; Nes, I.F.; Abee, T.; Konings, W.N.

and Venema, G. (1993) Appl. Environ. Microbiol., 59(11), 3577-3584.

[56] Chen, Y.; Shapira, R.; Eisenstein, M. and Montville, T.J. (1997) Appl. Environ. Microbiol., 63(2), 524-531.

[57] Montville, T.J. and Chen, Y. (1998) Appl. Microbiol. Biotechnol., 50(5), 511-519.

[58] Herranz, C.; Chen, Y.; Chung, H.C.; Cintas, L.M.; Hernández, P.E.; Montville, T.J. and Chikindas, M.L. (2001) Appl. Environ.

Microbiol., 67(4), 1689-1692. [59] Herranz, C.; Cintas, L.M.; Hernández, P.E.; Moll, G.N. and Dries-

sen, A.J. (2001) Antimicrob. Agents Chemother., 45(3), 901-904. [60] Bruno, M.E.C. and Montville, T.J. (1993) Appl. Environ. Micro-

biol., 59(9), 3003-3010. [61] Kaiser, A.L. and Montville, T.J. (1996) Appl. Environ. Microbiol.,

62(12), 4529-4535. [62] Héchard, Y. and Sahl, H.-G. (2002) Biochimie, 84(5-6), 545-557.

[63] Suzuki, M.; Yamamoto, T.; Kawai, Y.; Inoue, N. and Yamazaki, K. (2005) J. Appl. Microbiol., 98(5), 1146-1151.

[64] Fimland, G.; Johnsen, L.; Axelsson, L.; Brurberg, M.B.; Nes, I.F.; Eijsink, V.G.H. and Nissen-Meyer, J. (2000) J. Bacteriol., 182(9),

2643-2648. [65] Johnsen, L.; Fimland, G.; Eijsink, V. and Nissen-Meyer, J. (2000)

Appl. Environ. Microbiol., 66(11), 4798-4802. [66] Fimland, G.; Eijsink, V.G.H. and Nissen-Meyer, J. (2002) Bio-

chemistry, 41(30), 9508-9515.

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 35

[67] Fregeau, G.N.L.; Sailer, M.; Niemczura, W.P.; Nakashima, T.T.;

Stiles, M.E. and Vederas, J.C. (1997) Biochemistry, 36(49), 15062-15072.

[68] Wang, Y.; Henz, M.E.; Gallagher, F.N.L.; Chai, S.; Gibbs, A.C.; Yan, L.Z.Y.; Stiles, M.E.; Wishart, D.S. and Vederas, J.C. (1999)

Biochemistry, 38(47), 15438-15447. [69] Haugen, H.S.; Fimland, G.; Nissen-Meyer, J. and Kristiansen, P. E.

(2005) Biochemistry, 44(49), 16149-16157. [70] Uteng, M.; Hauge, H.H.; Markwick, P.R.L.; Fimland, G.; Mantzi-

las, D.; Nissen-Meyer, J. and Muhle-Goll, C. (2003) Biochemistry, 42(39), 11417-11426.

[71] Watson, R.M.; Woody, R.W.; Lewis, R.V.; Bohle, D.S.; Andreotti, A.H.; Ray, B. and Miller, K.W. (2001) Biochemistry, 40(46),

14037-14046. [72] Chen, Y.; Ludescher, R.D. and Montville, T.J. (1997) Appl. Envi-

ron. Microbiol., 63(12), 4770-4777. [73] Kazazic, M.; Nissen-Meyer, J. and Fimland, G. (2002) Microbiol-

ogy, 148(7), 2019-2027. [74] Morisset, D.; Berjeaud, J.-M.; Marion, D.; Lacombe, C. and Frere,

J. (2004). Appl. Environ. Microbiol., 70(8), 4672-4680. [75] Miller, K.W.; Schamber, R.; Osmanagaoglu, O. and Ray, B. (1998)

Appl. Environ. Microbiol., 64(6), 1997-2005. [76] Fimland, G.; Pirneskoski, J.; Kaewsrichan, J.; Jutila, A.; Kristian-

sen, P.E.; Kinnunen, P.K.J. and Nissen-Meyer, J. (2006) Biochim. Biophys. Acta, 1764(6), 1132-1140.

[77] Miller, K.W.; Schamber, R.; Chen, Y. and Ray, B. (1998) Appl. Environ. Microbiol., 64(1), 14-20.

[78] Killian, J.A. and von Heijne, G. (2000) Trends Biochem. Sci., 25(9), 429-434.

[79] Johnsen, L.; Fimland, G. and Nissen-Meyer, J. (2005) J. Biol. Chem., 280(10), 9243-9250.

[80] Ramnath, M.; Beukes, M.; Tamura, K. and Hastings, J.W. (2000) Appl. Environ. Microbiol., 66(7), 3098-3101.

[81] Gravesen, A.; Warthoe, P.; Knøchel, S. and Thirstrup, K. (2000) Microbiology, 146(6), 1381-1389.

[82] Héchard, Y.; Pelletier, C.; Cenatiempo, Y. and Frère, J. (2001) Microbiology, 147(6), 1575-1580.

[83] Gravesen, A.; Ramnath, M.; Rechinger, K.B.; Andersen, N.; Jänsch, L.; Héchard, Y.; Hastings, J.W. and Knøchel, S. (2002) Mi-

crobiology, 148(8), 2361-2369. [84] Dalet, K.; Cenatiempo, Y.; Cossart, P.; Héchard, Y.; European

Listeria Genome Consortium (2001) Microbiology, 147(12), 3263-3269.

[85] Ramnath, M.; Arous, S.; Gravesen, A.; Hastings, J.W. and Hé-chard, Y. (2004) Microbiology, 150(8), 2663-2668.

[86] Xue, J.; Hunter, I.; Steinmetz, T.; Peters, A.; Ray, B. and Miller, K.W. (2005) Appl. Environ. Microbiol., 71(3), 1283-1290.

[87] Diep, D.B.; Skaugen, M.; Salehian, Z.; Holo, H. and Nes, I.F. (2007) Proc. Natl. Acad. Sci. USA, 104(7), 2384-2389.

[88] Fimland, G.; Jack, R.; Jung, G.; Nes, I.F. and Nissen-Meyer, J. (1998) Appl. Environ. Microbiol., 64(12), 5057-5060.

[89] Saavedra, L.; Minahk, C.; de Ruiz Holgado, A.P. and Sesma, F. (2004) Antimicrob. Agents. Chemother., 48(7), 2778-2781.

[90] Yan, L.Z.; Gibbs, A.C.; Stiles, M.E.; Wishart, D.S. and Vederas, J.C. (2000) J. Med. Chem., 43(24), 4579-4581.

[91] Kaur, K.; Andrew, L.C.; Wishart, D.S. and Vederas, J.C. (2004) Biochemistry, 43(28), 9009-9020.

[92] Venema, K.; Kok, J.; Marugg, J.D.; Toonen, M.Y.; Ledeboer, A.M.; Venema, G. and Chikindas, M.L. (1995) Mol. Microbiol.,

17(3), 515-522. [93] Quadri, L.E.N.; Kleerebezem, M.; Kuipers, O.P.; De Vos, W.M.;

Roy, K.L.; Vederas, J.C. and Stiles, M.E. (1997) J. Bacteriol., 179(19), 6163-6171.

[94] Quadri, L.E.N.; Sailer, M.; Terebiznik, M.R.; Roy, K.L.; Vederas, J.C. and Stiles, M.E. (1995) J. Bacteriol., 177(5), 1144-1151.

[95] Dayem, M.A.; Fleury, Y.; Devilliers, G.; Chaboisseau, E.; Girard, R.; Nicolas, P. and Delfour, A. (1996) FEMS Microbiol. Lett.,

138(2-3), 251-259. [96] Fimland, G.; Eijsink, V.G.H. and Nissen-Meyer, J. (2002) Micro-

biology, 148(11), 3661-3670. [97] Johnsen, L.; Fimland, G.; Mantzilas, D. and Nissen-Meyer, J.

(2004) Appl. Environ. Microbiol., 70(5), 2647-2652. [98] Sprules, T.; Kawulka, K.E. and Vederas, J.C. (2004) Biochemistry,

43(37), 11740-11749. [99] Dalhus, B.; Johnsen, L. and Nissen-Meyer J. (2003) Acta Cryst.,

D59(7), 1291-1293.

[100] Johnsen, L.; Dalhus, B.; Leiros, I. and Nissen-Meyer, J. (2005) J.

Biol. Chem., 280(19), 19045-19050. [101] Kim, I.-K.; Kim, M.-K.; Kim, J.-H.; Yim, H.-S.; Cha, S.-S. and

Kang, S.-O. (2007) BMC Struct. Biol., 7, 35. [102] Sprules, T.; Kawulka, K.E.; Gibbs, A.C.; Wishart, D.S. and Ved-

eras, J.C. (2004) Eur. J. Biochem., 271(9), 1748-1756. [103] Håvarstein, L.S.; Diep, D.P. and Nes, I.F. (1995) Mol. Microbiol.,

16(2), 229-240. [104] Håvarstein, L.S.; Holo, H. and Nes, I.F. (1994) Microbiology,

140(9), 2383-2389. [105] van Belkum, M.J.; Worobo, R.W. and Stiles, M.E. (1997) Mol.

Microbiol., 23(6), 1293-1301. [106] Aucher, W.; Lacombe, C.; Héquet, A.; Frère, J. and Berjeaud, J.-M.

(2005). J. Bacteriol., 187(6), 2218-2223. [107] van der Meer, J.R.; Rollema, H.S.; Siezen, R.J.; Beerthuyzen,

M.M.; Kuipers, O.P. and de Vos, W.M. (1994) J. Biol. Chem., 269(5), 3555-3562.

[108] Ray, B.; Schamber, R. and Miller, K.W. (1999) Appl. Environ. Microbiol., 65(6), 2281-2286.

[109] Nissen-Meyer, J.; Holo, H.; Håvarstein, L.S.; Sletten, K. and Nes, I.F. (1992) J. Bacteriol., 174(17), 5686-5692.

[110] Franz, C.M.A.P.; Grube, A.; Herrmann, A.; Abriouel, H.; Stärke, J.; Lombardi, A.; Tauscher, B. and Holzapfel, W.H. (2002) Appl.

Environ. Microbiol., 68(5), 2550-2554. [111] Balla, E.; Dicks, L.M.T.; Du Toit, M.; van der Merwe, M.J. and

Holzapfel, W.H. (2000) Appl. Environ. Microbiol., 66(4), 1298-1304.

[112] Balla, E. and Dicks, L.M.T. (2005) Int. J. Food Microbiol., 99(1), 33-45.

[113] Zendo, T.; Koga, S.; Shigeri, Y.; Nakayama, J. and Sonomoto, K. (2006) Appl. Environ. Microbiol., 72(5), 3383-3389.

[114] Anderssen, E.L.; Diep, D.B.; Nes, I.F.; Eijsink, V.G.H. and Nissen-Meyer, J. (1998) Appl. Environ. Microbiol., 64(6), 2269-2272.

[115] Diep, D.B.; Håvarstein, L.S. and Nes, I.F. (1996) J. Bacteriol., 178(15), 4472-4483.

[116] Jiménez-Diaz, R.; Ruiz-Barba, J.L.; Cathcart, D.P.; Holo, H.; Nes, I.F.; Sletten, K.H. and Warner, P.J. (1995) Appl. Environ. Micro-

biol., 61(12), 4459-4463. [117] Stephens, S.K.; Floriano, B.; Cathcart, D.P.; Bayley, S.A.; Witt,

V.F.; Jiménez-Díaz, R.; Warner, P.J. and Ruiz-Barba, J.L. (1998) Appl. Environ. Microbiol., 64(5), 1871-1877.

[118] Maldonado, A.; Ruiz-Barba, J.L. and Jiménez-Diaz, R. (2003) Appl. Environ. Microbiol., 69(1), 383-389.

[119] Allison, G.E.; Fremaux, C. and Klaenhammer, T.R. (1994) J. Bac-teriol., 176(8), 2235-2241.

[120] Fremaux, C.; Ahn, C. and Klaenhammer, T.R. (1993) Appl. Envi-ron. Microbiol., 59(1), 3906-3915.

[121] McCormick, J.K.; Poon, A.; Sailer, M.; Gao, Y.; Roy, K.L.; McMullen, L.M.; Vederas, J.C.; Stiles, M.E. and van Belkum, M.J.

(1998) Appl. Environ. Microbiol., 64(12), 4757-4766. [122] Marciset, O.; Jeronimus-Stratingh, M.C.; Mollet, B. and Poolman,

B. (1997) J. Biol. Chem., 272(22), 14277-14284. [123] van Belkum, M.J.; Hayema, B.J.; Jeeninga, R.E.; Kok, J. and Ve-

nema, G. (1991) Appl. Environ. Microbiol., 57(2), 492-498. [124] Flynn, S.; van Sinderen, D.; Thornton, G.M.; Holo, H.; Nes, I.F.

and Collins, J.K. (2002) Microbiology, 148(4), 973-984. [125] Barrett, E.; Hayes, M.; O’Connor, P.; Gardiner, G.; Fitzgerald,

G.F.; Stanton, C.; Ross, R.P. and Hill, C. (2007) Appl. Environ. Microbiol., 73(11), 3719-3723.

[126] Qi, F.; Chen, P. and Caufield, P.W (2001) Appl. Environ. Micro-biol., 67(1), 15-21.

[127] Cuozzo, S.A.; Sesma, F.; Palacios, J.M.; de Ruiz Holgado, A.P. and Raya, R.R. (2000) FEMS Microbiol. Lett., 185(2), 157-161.

[128] Contreras, B.G.L.; De Vuyst, L.; Devreese, B.; Busanyova, K.; Raymaeckers, J.; Bosman, F.; Sablon, E. and Vandamme, E.J.

(1997) ) Appl. Environ. Microbiol., 63(1), 13-20. [129] Majhenic, A.C.; Venema, K.; Allison, G.E.; Matijasic, B.B.; Ro-

gelj, I. and Klaenhammer, T.R. (2004) Appl. Microbiol. Biotech-nol., 63(6), 705-714.

[130] Blom, H.; Katla, T.; Holck, A.; Sletten, K.; Axelsson, L. and Holo, H. (1999) Curr. Microbiol., 39(1), 43-48.

[131] Cintas, L.M.; Casaus, P.; Holo, H.; Hernandez, P.E.; Nes, I.F. and Håvarstein, L.S. (1998) J. Bacteriol., 180(8), 1988-1994.

[132] Ghrairi, T.; Frére, J.; Berjeaud, J.M. and Manai, M. (2005) Int. J. Food Microbiol., 105(3), 389-398.

36 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 Nissen-Meyer et al.

[133] Moll, G.; Ubbink-Kok, T.; Hauge, H.H.; Nissen-Meyer, J.; Nes,

I.F.; Konings, W.N. and Driessen, A.J.M. (1996) J. Bacteriol., 178(3), 600-605.

[134] Moll, G.; Hauge, H.H.; Nissen-Meyer, J.; Nes, I.F.; Konings, W.N. and Driessen, A.J.M. (1998) J. Bacteriol., 180(1), 96-99.

[135] Moll, G.N.; van den Akker, E.; Hauge, H.H.; Nissen-Meyer, J.; Nes, I.F.; Konings, W.N. and Driessen, A.J.M. (1999) J. Bacteriol.

181(16), 4848-4852. [136] Abee, T.; Klaenhammer, T.R. and Letellier, L. (1994) Appl. Envi-

ron. Microbiol., 60(3), 1006-1013. [137] Cuozzo, S.A.; Castellano, P.; Sesma, F.J.M.; Vignolo, G.M. and

Raya, R.R. (2003) Curr. Microbiol., 46(3), 180-183. [138] Nes, I.F.; Håvarstein, L.S. and Holo, H. (1995) in Genetics of

Streptococci, Enterococci and Lactococci, Developments in Bio-logical Standards (Ferretti, J.J.; Gilmore, M.S.; Klaenhammer, T.R.

and Brown, F., Eds.). Karger, Basel, Vol. 85, pp. 645-651. [139] Oppegård, C.; Fimland, G.; Thorbæk, L. and Nissen-Meyer, J.

(2007) Appl. Environ. Microbiol., 73(9), 2931-2938. [140] Hauge, H.H.; Nissen-Meyer, J.; Nes, I.F. and Eijsink, V.G.H.

(1998) Eur. J. Biochem., 251(3), 565-572. [141] Hauge, H.H.; Mantzilas, D.; Eijsink, V.G.H. and Nissen-Meyer, J.

(1999) J. Bacteriol., 181(3), 740-747. [142] Rogne, P.; Fimland, G.; Nissen-Meyer, J. and Kristiansen, P.E.

(2008) Biochim. Biophys. Acta, 1784, 543-554. [143] Fimland, N.; Fimland, G.; Rogne, P.; Nissen-Meyer, J. and Kris-

tiansen, P.E. (2008) Biochim. Biophys. Acta. in press. [144] Senes, A.; Engel, D.E. and DeGrado, W.F. (2004) Curr. Opin.

Struct. Biol., 14(4), 465-479. [145] Garneau, S.; Ference, C.A.; van Belkum, M.J.; Stiles, M.E. and

Vederas, J.C. (2003) Appl. Environ. Microbiol., 69(3), 1352-1358. [146] Maqueda, M.; Sánchez-Hidalgo, M.; Fernández, M.; Montalbán-

Lopez, M.; Valdivia, E. and Martínez-Bueno, M. (2008) FEMS Mi-crobiol. Rev., 32(1), 2-22.

[147] Franz, C.A.P.; van Belkum, M.J.; Holzapfel, W.H.; Abriouel, H. and Gálvez, A. (2007) FEMS Microbiol. Rev., 31(3), 293-310.

[148] Kemperman, R.; Kuipers, A.; Karsens, H.; Nauta, A.; Kuipers, O. and Kok, J. (2003) Appl. Environ. Microbiol., 69(3), 1589-1597.

[149] Martinez-Bueno, M.; Maqueda, M.; Gálvez, A.; Samyn, B.; van Beeumen, J.; Coyette, J. and Valdivia, E. (1994) J. Bacteriol.,

176(20), 6334-6339. [150] Martinez-Bueno, M.; Valdivia, E.; Gálvez, A.; Coyette, J. and

Maqueda, M. (1998) Mol. Microbiol., 27(2), 347-358. [151] Maqueda, M.; Gálvez, A.; Martinez-Bueno, M.; Sanchez-Barrena,

M.J.; González, C.; Albert, A.; Rico, M. and Valdivia, E. (2004) Curr. Protein Pept. Sci., 5(5), 399-416.

[152] Kawai, Y.; Saito, T.; Kitazawa, H. and Itoh, T. (1998) Biosci. Bio-technol. Biochem., 62(12), 2438-2440.

[153] Kawai, Y.; Saito, T.; Suzuki, M. and Itoh, T. (1998) Biosci. Bio-technol. Biochem., 62(5), 887-892.

[154] Kawai, Y.; Kemperman, R.; Kok, J. and Saiti, T. (2004) Curr. Protein Pept. Sci., 5(5), 393-398.

[155] Kawai, Y.; Ishii, Y.; Uemura, K.; Kitazawa, H.; Saito, T. and Itoh, T. (2001) Food Microbiol., 18(4), 407-415.

[156] Kawai, Y.; Ishii, Y.; Arakawa, K.; Uemura, K.; Saitoh, B.; Nishi-mura, J.; Kitazawa, H.; Yamazaki, Y.; Tateno, Y.; Itoh, T. and

Saito, T. (2004) Appl. Environ. Microbiol., 70(5), 2906-2911. [157] Kalmokoff, M.L. and Teather, R.M. (1997) Appl. Environ. Micro-

biol., 63(2), 394-402. [158] Kalmokoff, M.L.; Cyr, T.D.; Hefford, M.A.; Whitford, M.F. and

Teather, R.M. (2003) Can. J. Microbiol., 49(12), 763-773. [159] Kawulka, K.E.; Sprules, T.; Diaper, C.M.; Whittal, R.M.; McKay,

R.T.; Mercier, P.; Zuber, P. and Vederas, J.C. (2004) Biochemistry, 43(12), 3385-3395.

[160] Kawulka, K.; Sprules, T.; McKay, R.T.; Mercier, P.; Diaper, C.M.; Zuber, P. and Vederas, J.C. (2003) J. Am. Chem. Soc., 125(16),

4726-4727. [161] Leer, R.J.; van der Vossen, J.M.B.M.; van Giezen, M.; van Noort,

J.M. and Pouwels, P.H. (1995) Microbiology, 141(7), 1629-1635. [162] Wirawan, R.E.; Swanson K.M.; Kleffmann, T.; Jack, R.W. and

Tagg, J.R. (2007) Microbiology, 153(5), 1619-1630. [163] Deraz, S.F.; Hedström, M.; Nordberg Karlsson, E.; Linse, S.;

Khalil, A.A. and Mattiasson, B. (2007) World J. Microbiol. Bio-technol., 23(7), 911-921.

[164] Joosten, H.M.L.J.; Nunez, M.; Devreese, B.; van Beeumen, J. and Marugg, J.D. (1996) Appl. Environ. Microbiol., 62(11), 4220-4223.

[165] Maisnier-Patin, S.; Forni, E. and Richard, J. (1996) Int. Food Mi-

crobiol., 30(3), 255-270. [166] Tomita, H.; Fujimoto, S.; Tanimoto, K. and Ike, Y. (1997) J. Bac-

teriol., 179(24), 7843-7855. [167] Diaz, M.; Valdivia, E.; Martinez-Bueno, M.; Fernández, M.; Soler-

González, A.S.; Ramirez-Rodrigo, H. and Maqueda, M. (2003) Appl. Environ. Microbiol., 69(2), 1229-1236.

[168] González, C.; Langdon, G.M.; Bruix, M.; Gálvez, A.; Valdivia, E.; Maqueda, M. and Rico, M. (2000) Proc. Natl. Acad. Sci. USA,

97(21), 11221-11226. [169] Sánchez-Barrena, M.J.; Martinez-Ripoll, M.; Gálvez, A.; Valdivia,

E.; Maqueda, M.; Cruz, V. and Albert, A. (2003) J. Mol. Biol., 334(3), 541-549.

[170] Joosten, H.M.L.J.; Rodriguez, E. and Nunez, M. (1997) Lett. Appl. Microbiol., 24(1), 40-42.

[171] Folli, C.; Ramazzina, I.; Arcidiaco, P.; Stoppini, M. and Berni, R. (2003) FEMS Microbiol. Lett., 221(1), 143-149.

[172] Leuders, T.; Birkemo, G.A.; Nissen-Meyer, J.; Andersen, Ø. and Nes, I.F. (2005) Antimicrob. Agents Chemother., 49(6), 2399-2406.

[173] Holo, H.; Nilssen, Ø. and Nes, I.F. (1991) J. Bacteriol., 173(12), 3879-3887.

[174] Stoddard, G.W.; Petzel, J.P.; van Belkum, M.J.; Kok, J. and McKay, L.L. (1992) Appl. Environ. Micriobiol., 58(6), 1952-1961.

[175] Sánchez-Hidalgo, M.; Maqueda, M.; Gálvez, A.; Abriouel, H.; Valdivia, E. and Martinez-Bueno, M. (2003) Appl. Environ. Micro-

biol., 69(3), 1633-1641. [176] Casaus, P.; Nilsen, T.; Cintas, L.M.; Nes, I.F.; Hernández, P.E. and

Holo, H. (1997) Microbiology, 143(7), 2287-2294. [177] Holck, A.L.; Axelsson, L. and Schillinger, U. (1994) Curr. Micro-

biol., 29(2), 63-68. [178] Worobo, R.W.; Henkel, T.; Sailer, M.; Roy, K.L.; Vederas, J.C.

and Stiles, M.E. (1994) Microbiology, 140(3), 517-526. [179] Inoue, T.; Tomita, H. and Ike, Y. (2006) Antimicrob. Agents. Che-

mother., 50(4), 1202-1212. [180] Cintas, L.M.; Casaus, P.; Herranz, C.; Håvarstein, L.S.; Holo, H.;

Hernández, P.E. and Nes, I.F. (2000) J. Bacteriol., 182(23), 6806-6814.

[181] Floriano, B.; Ruiz-Barba, J.L. and Jiménez-Diaz, R. (1998) Appl. Environ. Microbiol., 64(12), 4883-4890.

[182] Martín-Platero, A.M.; Valdivia, E.; Ruiz-Roriguez, M.; Soler, J.J.; Martin-Vivaldi, M.; Maqueda, M. and Martinez-Bueno, M. (2006)

Appl. Environ. Microbiol., 72(6), 4245-4249. [183] Yamamoto, Y.; Togawa, Y.; Shimosaka, M. and Okazaki, M.

(2003) Appl. Environ. Microbiol., 69(10), 5746-5753. [184] Netz, D.J.A.; Sahl, H.-G.; Marcolino, R.; Nascimento, J.S.;

Oliveira, S.S.; Soares, M.B. and Bastos, M.C.F. (2001) J. Mol. Biol., 311(5), 939-949.

[185] Netz, D.J.A.; Pohl, R.; Beck-Sickinger, A.G.; Selmer, T.; Pierik, A.J.; Bastos, M.C.F. and Sahl, H.-G. (2002) J. Mol. Biol., 319(3),

745-756. [186] Fujita, K.; Ichimasa, S.; Zendo, T.; Koga, S.; Yoneyama, F.; Naka-

yama, J. and Sonomoto, K. (2007) Appl. Environ. Microbiol., 73(9), 2871-2877.

[187] Iwatani, S.; Zendo, T.; Yoneyama, F.; Nakayama, J. and Sonomoto, K. (2007) Biosci. Biotechnol. Biochem., 71(8), 1984-1992.

[188] Hyink, O.; Balakrishnan, M. and Tagg, J.R. (2005) FEMS Micro-biol. Lett., 252(2), 235-241.

[189] Venema, K.; Abee, T.; Haandrikman, A.J.; Leenhouts, K.J.; Kok, J.; Konings, W.N. and Venema, G. (1993) Appl. Environ. Micro-

biol., 59(4), 1041-1048. [190] Kanatani, K.; Oshimura, M. and Sano, K. (1995) Appl. Environ.

Microbiol., 61(3), 1061-1067. [191] Stern, N.J.; Svetoch, E.A.; Eruslanov, B.V.; Perelygin, V.V.; Mit-

sevich, E.V.; Mitsevich, I.P.; Pokhilenko, V.D.; Levchuk, V.P.; Svetoch, O.E. and Seal, B.S. (2006) Antimicrob. Agents Che-

mother., 50(9), 3111-3116. [192] Holck, A.; Axelsson, L. and Schillinger, U. (1996) FEMS Micro-

biol. Lett., 136(2), 163-168. [193] Srionnual, S.; Yanagida, F.; Lin, L.-H.; Hsiao, K.-N. and Chen, Y.-

S. (2007) Appl. Environ. Microbiol., 73(7), 2247-2250. [194] Han, K.-S.; Kim, Y.; Kim, S.-H. and Oh, S. (2007) J. Microbiol.

Biotechnol., 17(5), 774-783. [195] Chumchalova, J.; Stiles, J.; Josephsen, J. and Plockova, M. (2004)

J. Appl. Microbiol., 96(5), 1082-1089. [196] Tahara, T. and Kanatani, K. (1997) FEMS Microbiol. Lett., 147(2),

287-290.

Structure-Function Relationships of the Non-Lanthionine-Containing Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1 37

[197] Yarmus, M.; Mett, A. and Shapira, R. (2000) Biochem. Biophys.

Acta, 1490(3), 279-290. [198] Kim, M.-H.; Kong, Y-J.; Baek, H. and Hyun, H.-H. (2005) J. Food

Prot., 68(1), 157-163. [199] Gajic, O.; Buist, G.; Kojic, M.; Topisirovic, L.; Kuipers, O.P. and

Kok, J. (2003) J. Biol. Chem., 278(36), 34291-34298. [200] Revol-Junelles, A.M; Mathis, R.; Krier, F.; Fleury, Y; Delfour, A.

and Lefebvre, G. (1996) Lett. Appl. Microbiol., 23(2), 120-124. [201] Héchard, Y.; Berjeaud, J.-M. and Cenatiempo, Y. (1999) Curr.

Microbiol., 39(5), 265-269. [202] Revol-Junelles, A.-M. and Lefebvre, G. (1996) Curr. Microbiol.,

33(2), 136-137. [203] Papathanasopoulos, M.A.; Dykes, G.A.; Revol-Junelles, A.-M.;

Delfour, A.; von Holy, A. and Hastings, J.W. (1998) Microbiology, 144(5), 1343-1348.

[204] Martínez, B.; Fernández, M.; Suárez, J.E. and Rodriguez, A. (1999) Microbiology, 145(3), 3155-3161.

[205] Whitford, M.F.; McPherson, M.A.; Forster, R. J. and Teather, R.M. (2001) Appl. Environ. Microbiol., 67(2), 569-574.

[206] Tahara, T. and Kanatini, K. (1997) Biosci. Biotech. Biochem., 61(5), 884-886.

[207] Tahara, T.; Oshimura, M.; Umezawa, C. and Kanatani, K. (1996) Appl. Environ. Microbiol., 62(3), 892-897.

[208] Tahara, T.; Yoshioka, S.; Utsumi, R. and Kanatani, K. (1997) FEMS Microbiol. Lett., 148(1), 97-100.

[209] van Belkum, M.J.; Kok, J.; Venema, G.; Holo, H.; Nes, I.F.; Kon-

ings, W.N. and Abee, T. (1991) J. Bacteriol., 173(24), 7934-7941. [210] Nissen-Meyer, J.; Håvarstein, L.S.; Holo, H.; Sletten, K. and Nes,

I.F. (1993) J. Gen. Microbiol., 139(7), 1503-1509. [211] Venema, K.; Haverkort, R.E.; Abee, T.; Haandrikman, A.J.; Leen-

houts, K.J.; de Leij, L.; Venema, G. and Kok, J. (1994) Mol. Mi-crobiol., 14(3), 521-532.

[212] Gálvez, A.; Valdivia, E.; Abriouel, H.; Camafeita, E.; Mendez, E.; Martinez-Bueno, M. and Maqueda, M. (1998) Arch. Microbiol.,

171(1), 59-65. [213] Netz, D.J.A.; Bastos, M.C.F. and Sahl, H.-G. (2002) Appl. Environ.

Microbiol., 68(11), 5274-5280. [214] Venema, K.; Dost, M.H.R; Venema, G. and Kok, J. (1996) Micro-

biology, 142(10), 2825-2830. [215] Remiger, A.; Eijsink, V.G.H.; Ehrmann, M.A.; Sletten, K.; Nes,

I.F. and Vogel, R.F. (1999) J. Appl. Microbiol., 86(6), 1053-1058. [216] Benoit, V.; Lebrihi, A; Milliére, J.-B. and Lefebvre, G. (1997)

Curr. Microbiol., 34(3), 173-179. [217] Callewaert, R.; Holo, H.; Devreese, B.; Van Beeumen, J.; Nes, I.

and De Vuyst, L. (1999) Microbiology, 145(9), 2559-2568. [218] Ehrmann, M.A.; Remiger, A.; Eijsink, V.G.H. and Vogel, R.F.

(2000) Biochem. Biophys. Acta, 1490(3), 355-361. [219] Quadri, L.E.N.; Yan, L.Z.; Stiles, M.E. and Vederas, J.C. (1997) J.

Biol. Chem., 272(6), 3384-3388. [220] Tominaga, T. and Hatakeyama, Y. (2006) Appl. Environ. Micro-

biol., 72(2), 1141-1147.

Received: February 02, 2008 Revised: May 19, 2008 Accepted: May 19, 2008